CN104080912A

CN104080912A - Combination therapy for treating hearing and balance disorders

Info

Publication number: CN104080912A
Application number: CN201380007519.6A
Authority: CN
Inventors: 斯维特拉娜·亚当斯基; 埃琳娜·费因斯坦
Original assignee: Quark Pharmaceuticals Inc
Current assignee: Quark Pharmaceuticals Inc
Priority date: 2012-01-12
Filing date: 2013-01-10
Publication date: 2014-10-01
Also published as: WO2013106494A1; IL233141B; US20150126586A1; KR20140111673A; EP2802657B1; IL233141A0; US20140364484A1; AU2013208012A1; CA2858630A1; BR112014016937A2; JP2016528161A; ZA201404268B; US9434946B2; EP2802657A1; RU2014125496A

Abstract

The present application relates to combinations of inhibitors directed at down-regulation of genes associated with hearing loss including HES1, HES5, HEY2, CDKN1B and NOTCH1, exhibiting a beneficial effect and useful in treating or attenuating hearing loss, treating balance impairment, promoting the replacement, regeneration, or protection of otic (sensory) hair cells of the inner ear, and or effecting hearing restoration / regeneration.

Description

Be used for the treatment of the combination treatment of hearing and disequilibrium

Related application

The application requires the name that on January 12nd, 2012 submits to be called " Compounds, Compositions and Methods For Treating Hearing Loss " U.S. Provisional Application number 61/585, the name of submitting on August 3rd, 672 and 2012 is called the rights and interests of the PCT application number PCT/US12/49616 of " Double-Stranded Oligonucleotide Compounds and Methods of Use Thereof for Treating Hearing and Balance Disorders ", these patent applications entirety and being incorporated herein by reference for all objects.

Sequence table

The application comprises Nucleotide and/or aminoacid sequence with way of reference, and these sequences are present in name that size creates with IBM-PC machine form (operating system and MS-Windows compatibility) for 5.044MB and on January 9th, 2013 and that together submit to therewith and are called in the file of " 237-PCT2.ST25.txt ".

Technical field

The disclosure relates to and can be used for treating hearing loss, and treatment disequilibrium, promotes replacement, regeneration or the protection of inner ear tragus (sensation) cell, or realizes the combination treatment of auditory rehabilitation/regeneration, comprises composition and method.

Background of invention

The pharmaceutical composition that the PCT application number PCT/US12/49616 that authorizes the application's transferee relates to double-stranded RNA compound, comprise described compound and the using method aspect the downward target gene relevant to hearing loss and disequilibrium (comprising HES1, HES5, HEY1, HEY2, ID1, ID2, ID3, CDKN1B and NOTCH1) thereof; the inhibition of described gene can be used for treating hearing loss; treatment disequilibrium; promote replacement, regeneration or the protection of inner ear tragus (sensation) cell, or realize auditory rehabilitation/regeneration.

Brief summary of the invention

Have now found that, relate to some three joint group of the inhibitor that relevant to hearing and disequilibrium some target gene lowers are closed be of value to treatment or alleviate hearing loss, treatment disequilibrium, promote inner ear ear (sensation) hair cell replacement, regeneration or protection and/or realize auditory rehabilitation/regeneration.Specifically, the 3rd medicament of the first medicament that this combination comprises target HES1, the second medicament of target HES5 and target HEY2, or the first medicament that comprises target CDKN1B, the second medicament of target NOTCH1 and the 3rd medicament of target HEY2.

The combination of a kind of HES1 inhibitor for therapy, HES5 inhibitor and HEY2 inhibitor is provided in one aspect, herein.In some embodiments, this therapy comprises prevention, treatment or postpones the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevents ear (sensation) hair cell loss of inner ear.On the other hand, provide a kind of composition herein, the combination that it comprises HES1 inhibitor or its pharmacy acceptable salt or prodrug, HES5 inhibitor or its pharmacy acceptable salt or prodrug and HEY2 inhibitor or its pharmacy acceptable salt or prodrug; And pharmaceutically acceptable carrier.Aspect another, a kind of prevention, treatment are provided herein or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent from the method for ear (sensation) hair cell loss of inner ear from comprising to experimenter and using HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor.On the other hand, provide a kind of product, test kit or commercial package herein, it comprises HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor or its composition as disclosed.

Aspect another, provide the combination of a kind of CDKN1B inhibitor for therapy, NOTCH1 inhibitor and HEY2 inhibitor herein.In some embodiments, this therapy comprises prevention, treatment or postpones the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevents ear (sensation) hair cell loss of inner ear.On the other hand, a kind of composition is provided herein, and it comprises at least one CDKN1B inhibitor or its pharmacy acceptable salt or prodrug, at least one NOTCH1 inhibitor or its pharmacy acceptable salt or prodrug and at least one HEY2 inhibitor or its pharmacy acceptable salt or prodrug; And pharmaceutically acceptable carrier.Aspect another, a kind of prevention, treatment are provided herein or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent from the method for ear (sensation) hair cell loss of inner ear from comprising to experimenter and using CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor.On the other hand, provide a kind of product, test kit or commercial package herein, it comprises CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor or its composition as disclosed.

In the many aspects and embodiment of combination provided in this article and method, every kind of inhibitor for the treatment of significant quantity is used substantially side by side, individually or in order and with any order, and these components are used individually or for example, as fixing combination (, in single formulation).In some embodiments of combination provided in this article and method, every kind of inhibitor side by side or in order and with any order is used substantially.In other embodiments of combination provided in this article and method, two kinds of inhibitor for the treatment of significant quantity side by side or are substantially side by side used, and the third inhibitor is used individually.

In one embodiment, the combination of a kind of HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor is provided herein, side by side or in order for preventing, treat or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent ear (sensation) hair cell loss of inner ear for substantially.

In another embodiment, the combination of a kind of CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor is provided herein, side by side or in order for preventing, treat or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent ear (sensation) hair cell loss of inner ear for substantially.

In one embodiment, be provided for treatment (comprising prevention) generation of experimenter's hearing loss or the method for seriousness, in experimenter, HES1, HES5 are with the expression of HEY2 gene and the nosetiology of dysaudia/hearing loss or make progress relevant.

In another embodiment, be provided for treatment (comprising prevention) generation of experimenter's dysaudia or the method for seriousness, in experimenter, HES1, HES5 are with the expression of HEY2 gene and the nosetiology of dysaudia or make progress relevant.

In another embodiment, be provided for the generation of ear (sensation) hair cell loss for the treatment of (comprising prevention) experimenter's inner ear or the method for seriousness, in experimenter, the expression of HES1, HES5 and HEY2 gene is relevant to nosetiology or the progress of ear (sensation) hair cell loss.

In one embodiment, be provided for treatment (comprising prevention) generation of experimenter's hearing loss or the method for seriousness, in experimenter, CDKN1B, NOTCH1 are with the expression of HEY2 gene and the nosetiology of dysaudia/hearing loss or make progress relevant.

In another embodiment, be provided for treatment (comprising prevention) generation of experimenter's dysaudia or the method for seriousness, in experimenter, CDKN1B, NOTCH1 are with the expression of HEY2 gene and the nosetiology of dysaudia or make progress relevant.

In another embodiment, be provided for the generation of ear (sensation) hair cell loss for the treatment of (comprising prevention) experimenter's inner ear or the method for seriousness, in experimenter, the expression of CDKN1B, NOTCH1 and HEY2 gene is relevant to nosetiology or the progress of ear (sensation) hair cell loss.

In one embodiment, a kind of prevention, treatment are provided herein or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent from the method for ear (sensation) hair cell loss of inner ear from comprising to experimenter and using HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor.

In another embodiment, a kind of prevention, treatment are provided herein or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent from the method for ear (sensation) hair cell loss of inner ear from comprising to experimenter and using CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor.

In some embodiments, experimenter is Mammals.In preferred embodiments, experimenter is human experimenter.

Prevention experimenter is also provided the method that auditory nerve is degenerated, comprises to experimenter and use combination disclosed herein.

These class methods relate to combination or the composition of the HES1 inhibitor, HES5 inhibitor and the HEY2 inhibitor that have the administration of needs to comprise prevention or treatment significant quantity to this treatment.In another embodiment, these class methods relate to combination or the composition of the CDKN1B inhibitor, NOTCH1 inhibitor and the HEY2 inhibitor that have the administration of needs to comprise prevention or treatment significant quantity to this treatment.

In multiple embodiments, every kind of inhibitor (HES1 inhibitor, HES5 inhibitor, HEY2 inhibitor, CDKN1B inhibitor and NOTCH1 inhibitor) is independently selected from the group being made up of little organic molecule, albumen, antibody or its fragment, peptide, plan peptide and nucleic acid molecule.

In preferred embodiments, every kind of inhibitor comprises therapeutic nucleic acid molecule or its pharmacy acceptable salt.In some embodiments, nucleic acid compound is applied directly to the round window membrane of cochlea, or by through injection of tympanum or via using through tympanum device (such as intubate or implant).Method comprises and continues to send and control is sent with part or systemic delivery, comprises that the implant that uses for example pump, slowly-releasing or sustained-release composition or comprise drug-reservoir sends inhibitor.

In the preferred embodiment of composition provided in this article, combination, method, commercial package and test kit, every kind of inhibitor comprises independent nucleic acid molecule or its pharmacy acceptable salt.In some embodiments of composition provided in this article, combination, method, commercial package and test kit, nucleic acid molecule is connected to each other.In some embodiments of composition provided in this article, combination, method, commercial package and test kit, nucleic acid molecule forms annealing covalently bound in (RNAistar) at multi-arm.

In the preferred embodiment of composition provided in this article, combination, method, commercial package and test kit, every kind of inhibitor comprises nucleic acid compound.Preferably, nucleic acid compound is applied directly to the round window membrane of cochlea, or by through injection of tympanum or via using through tympanum device (such as intubate).

Therefore, composition provided in this article, combination, method, commercial package and test kit preferably relate to nucleic acid molecule (for example short interfering nucleic acid (siNA) that uses binding nucleotide sequence (such as mRNA sequence) or its part, short interfering rna (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA)), described nucleotide sequence or its part coding HES1, HES5, HEY2, CDKN1B or NOTCH1, for example be respectively mankind HES1, HES5, HEY2, mRNA encoding sequence (the SEQ ID NO:1 of CDKN1B or NOTCH1mRNA, 2, 10, 7 and 11), they are encoded respectively as SEQ ID NO:12, 13, 21, 18 and 22 exemplified one or more albumen or protein protomers.In certain preferred aspects, composition disclosed herein, combination, method, commercial package and test kit are lowered or are suppressed HES1, HES5 and HEY2, or the expression of CDKN1B, NOTCH1 and HEY2 gene.In multiple embodiments, each nucleic acid molecule is selected from lowers dsRNA compound unmodified or chemically modified that HES1, HES5, HEY2, CDKN1B or NOTCH1 express, such as siRNA or the shRNA of chemically modified.

In some preferred embodiments, HES1 inhibitor is to lower double-stranded RNA (dsRNA) compound that synthesize, chemically modified that HES1 expresses.In certain preferred aspects, " HES1 " refers to mankind HES1 gene.In some preferred embodiments, HES5 inhibitor is to lower double-stranded RNA (dsRNA) compound that synthesize, chemically modified that HES5 expresses.In certain preferred aspects, " HES5 " refers to mankind HES5 gene.In some preferred embodiments, HEY2 inhibitor is to lower double-stranded RNA (dsRNA) compound that synthesize, chemically modified that HEY2 expresses.In certain preferred aspects, " HEY2 " refers to mankind HEY2 gene.In some preferred embodiments, CDKN1B inhibitor is to lower double-stranded RNA (dsRNA) compound that synthesize, chemically modified that CDKN1B expresses.In certain preferred aspects, " CDKN1B " refers to mankind CDKN1B gene.In some preferred embodiments, NOTCH1 inhibitor is to lower double-stranded RNA (dsRNA) compound that synthesize, chemically modified that NOTCH1 expresses.In certain preferred aspects, " NOTCH1 " refers to mankind NOTCH1 gene.

In the certain preferred embodiments of composition provided herein, combination, method, commercial package and test kit, the first nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HES1 gene, the second nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HES5 gene, and the 3rd nucleic acid is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HEY2 gene.

In the certain preferred embodiments of composition provided herein, combination, method, commercial package and test kit, the first nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding CDKN1B gene, the second nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding NOTCH1 gene, and the 3rd nucleic acid is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HEY2 gene.

In the multiple embodiments of composition provided herein, combination, method, commercial package and test kit, each of double chain oligonucleotide (for example double-stranded RNA (dsRNA)) comprises sense strand and antisense strand.

In the certain preferred embodiments of composition provided herein, combination, method, commercial package and test kit, each of double chain oligonucleotide comprises sense strand and antisense strand, and wherein (a) every chain is that 18 to 49 Nucleotide are long independently; (b) the sequence complementation of the mRNA of the sequence of 18 of antisense strand to 49 Nucleotide and coding target gene; And (c) sequence of 18 of sense strand to 49 Nucleotide and antisense strand complementation.In multiple embodiments, the mRNA of coding target gene is selected from Mammals HES1 (SEQ ID NO:1), HES5 (SEQ ID NO:2), HEY2 (SEQ ID NO:10), CDKN1B (SEQ ID NO:7) or NOTCH1 (for example SEQ ID NO:11 or its part); And sense strand and antisense strand comprise SEQ ID NO:23-1495 or 26667-26706 (HES1), SEQ ID NO:1496-2703 or 26707-26732 (HES5), SEQ ID NO:13004-16621 or 26779-26788 (HEY2), SEQ ID NO:7444-10533 or 26867-26900 (CDKN1B) or SEQ ID NO:16622-26666 or 26901-26912 (NOTCH1) sequence pair shown in any one.

In the certain preferred embodiments of composition provided herein, combination, method, commercial package and test kit, the first double chain oligonucleotide comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

(b) the sequence complementation of the mRNA of the sequence of 18 of described antisense strand to 49 Nucleotide and coding HES1; And

(c) sequence of 18 of described sense strand to 49 Nucleotide and described antisense strand complementation;

The second double chain oligonucleotide comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

(b) the sequence complementation of the mRNA of the sequence of 18 of described antisense strand to 49 Nucleotide and coding HES5; And

(c) sequence of 18 of described sense strand to 49 Nucleotide and described antisense strand complementation; And

The 3rd double chain oligonucleotide comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

(b) the sequence complementation of the mRNA of the sequence of 18 of described antisense strand to 49 Nucleotide and coding HEY2; And

(c) sequence of 18 of described sense strand to 49 Nucleotide and described antisense strand complementation.

In other preferred embodiments of composition provided herein, combination, method, commercial package and test kit, the first double chain oligonucleotide is the dsRNA molecule that comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

(b) the sequence complementation of the mRNA of the sequence of 18 of described antisense strand to 49 Nucleotide and coding CDKN1B; And

(c) sequence of 18 of sense strand to 49 Nucleotide and antisense strand complementation; The second double chain oligonucleotide comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

(b) the sequence complementation of the mRNA of the sequence of 18 of described antisense strand to 49 Nucleotide and coding NOTCH1; And

(a) every chain is that 18 to 49 Nucleotide are long independently;

In some embodiments, dsRNA molecule has the structure shown in structure (A1) as disclosed herein or structure (A2).

In specific embodiments, composition provided herein, combination, method, commercial package and test kit can be used for treating ear's (ear, sense of hearing) illness or pathology, especially relate to the pathology of ear (sensation) Hair Cell Death of inner ear.

On the other hand, provide the purposes of the combination of HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor, for the medicament for the preparation for the treatment of inner ear or middle ear diseases or obstacle.On the other hand, provide the purposes of the combination of CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor, for the medicament for the preparation for the treatment of inner ear or middle ear diseases or obstacle.

In specific embodiments, combination, composition and the using method aspect the treatment sense of hearing and vestibular disease, obstacle, damage and illness thereof are provided herein, include but not limited to hearing loss that ear toxin causes, age related hearing loss, because the end-organ that relates to inner ear hair cells damages the dysaudia causing, for example sense of hearing wound, viral endolymphatic labyrinth inflammation, Meniere; Can be the tinnitus of intermittence or persistence, wherein diagnosis has phonosensitive dysacousis; The hearing loss causing due to bacterium or virus infection, such as in zoster oticus; The suppurative labyrinthitis being caused by acute otitis media, purulent meningitis, chronic otitis media, sudden deafness (comprising viral origin), the viral endolymphatic labyrinth inflammation for example being caused by virus, comprises parotitis, measles, influenza, varicella, mononucleosis and adenovirus; Congenital hearing loss, such as by rubella, when birth anoxic, cause into inner ear, erythroblastosis fetalis and heredity illness (Waardenburg's syndrome and gargoylism) because of bleeding of using to parent that wound during ototoxic drug causes.

On the other hand, provide the commercial package or the test kit that comprise any composition disclosed herein or combination herein.In some embodiments, this commercial package or test kit comprise label or specification sheets, and it provides about some information that can how to use composition disclosed herein or combination.In some embodiments of commercial package or test kit, label or specification sheets comprise drug administration information.In some embodiments of commercial package or test kit, label or specification sheets comprise using indicates.In some embodiments of commercial package or test kit, label or specification sheets indicate composition or combination is applicable to treatment.In some embodiments of commercial package or test kit, wherein label or specification sheets indicate composition or combination and are applicable to prevention, treatment or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, and/or prevent ear (sensation) hair cell loss of inner ear.

Be only exemplary by the preferred method being described, material and example now, be not intended to limit; Can be used for practice or inspection the present invention to those materials and methods similar or that be equal to as herein described.Other features and advantages of the present invention will be apparent by the following drawings, embodiment and claims.

Accompanying drawing summary

Figure 1A-1E has shown the ABR response obtaining after the 0th day, 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks in this research.

Figure 1A shown in this research the 0th day, 1KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.

Figure 1B shown in this research the 0th day, 4KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.

Fig. 1 C shown in this research the 0th day, 8KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.

Fig. 1 D shown in this research the 0th day, 16KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.

Fig. 1 E shown in this research the 0th day, 32KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.

Fig. 1 F provides the caption of Fig. 1 A – 1E.

Describe in detail

The composition of the expression of lowering some gene relevant to hearing loss and combination are provided herein and suffer from the purposes in the experimenter of hearing loss and/or disequilibrium in treatment, with promote inner ear the replacement of ear (sensation) hair cell, regenerate or protect and/or realize auditory rehabilitation/regeneration.In preferred embodiments, the method comprises partially or completely hearing regeneration.Show, the expression that suppresses HES1, HES5 and the combination of HEY2 gene or the combination of CDKN1B, NOTCH1 and HEY2 gene is of value to hearing regeneration.The application relates in particular to the purposes of for example double chain oligonucleotide molecule of therapeutical agent (comprising dsRNA/ siRNA (siRNA) compound) that suppresses HES1, HES5, CDKN1B, HEY2 and NOTCH1 expression and relates to the purposes of these dsRNA molecules in treatment hearing loss.Can be used for generating as herein provided the composition of dsRNA molecule and the sense strand of combination and complementary antisense strand at SEQ ID NO:23-1495 or 26667-26706 (HES1), SEQ ID NO:1496-2703 or 26707-26732 (HES5), SEQ ID NO:13004-16621 or 26779-26788 (HEY2), shown in SEQ ID NO:7444-10533 or 26867-26900 (CDKN1B) or SEQ ID NO:16622-26666 or 26901-26912 (NOTCH1).Some at present preferred sense strand and antisense strand to shown in following table I-V.

Be provided for suppressing in vivo HES1, HES5 and HEY2 genetic expression herein or for suppressing method, combination and the composition of CDKN1B, NOTCH1 and HEY2 genetic expression.In general, the method comprises being enough to use by the amount of RNA interference mechanism downward expression of target gene the combination/composition of oligoribonucleotide, such as the dsRNA molecule combination/composition of (comprising siRNA (being dsRNA)), described combination/composition target said target mrna is also hybridized with mRNA at biotic condition (in cell) or interacts, or uses the nucleic acid substances that can produce siRNA in cell.The details of target gene are shown in lower Table A.

Table A: target gene

Preferred target is HES1 (mRNA SEQ ID NO:1; Polypeptide SEQ ID NO:12), HES5 (mRNA SEQ ID NO:2; Polypeptide SEQ ID NO:13), HEY2 (mRNA SEQ ID NO:10; Polypeptide SEQ ID NO:21), CDKN1B (mRNA SEQ ID NO:7; Polypeptide SEQ ID NO:18) and NOTCH1 (mRNA SEQ ID NO:11; Polypeptide SEQ ID NO:22).

In multiple embodiments, provide the composition of double-stranded RNA (dsRNA) (comprising the siRNA (siRNA) of chemical modification)/the be combined in purposes for the treatment of various diseases and medical illness aspect.Specified disease to be treated and illness relate to hearing loss and/or balance loss.

Can be used for generating for the dsRNA of combination as herein provided, composition and method preferably have justice and anti sense nucleotide sequence is determined priority according to proprietary algorithm based on its scoring, using the optimal sequence as the expression of targeted human genoid.SEQ ID NO:23-693 and 26691-26706 (HES1), SEQ ID NO:1496-2029 and 26725-26732 (HES5), SEQ ID NO:7444-9007 and 26887-26900 (CDKN1B), SEQ ID NO:13004-14801 and 26785-26788 (HEY2), SEQ ID NO:16622-18643 and 26922 – 26912 (NOTCH1) show 19-aggressiveness oligomer.SEQ ID NO:694-1495 (HES1), SEQ ID NO:2030-2703 (HES5), SEQ ID NO:9008-10533 (CDKN1B), SEQ ID NO:14802-16389 (HEY2), SEQ ID NO:18644-26666 (NOTCH1) show the 18-aggressiveness oligomer that can be used for generating the dsRNA molecule that meets structure A2, as mentioned below.

definition

For simplicity, some term adopting in specification sheets, embodiment and claims will be described in herein.

It should be noted, as used herein, singulative " ", " one " and " being somebody's turn to do/described " also comprise plural form, unless pointed out clearly on the contrary.

In the time that each aspect of the present invention or embodiment are described according to Markush group or other alternative groupings, person of skill in the art will appreciate that therefore the present invention is also described according to any single member of this group or member's subgroup.

" inhibitor " is the compound that the activity decreased of the product of genetic expression or this gene can be enough to the degree that realizes required biology or physiological effect to (partially or completely).As used herein term " inhibitor " refer to following one or more: little organic molecule, albumen, antibody or its fragment, peptide, plan peptide and nucleic acid molecule, comprise siRNA, shRNA, synthetic shRNA, miRNA, sense-rna and DNA and ribozyme.

" dsRNA molecule " or " dsRNA inhibitor " is in the degree that is enough to realize required biology or physiological effect, to lower or to reduce the active compound of the product of genetic expression or this gene, and comprises one or more in siRNA, shRNA, synthetic shRNA, miRNA.Inhibition can also be called downward or for RNAi, be called silence.

Term " inhibition " refers to the activity decreased of the product of genetic expression or this gene to being enough to realize required biology or the degree of physiological effect as used herein.Inhibition can be completely or part.

As used herein, " inhibition " of term target gene means the genetic expression (transcribe or translate) of target gene or weakening, reduce or lowering of polypeptide active, and wherein target gene is selected from and is transcribed into SEQ ID NO:1,2,10,7 or 11 or the gene of SNP (single nucleotide polymorphism) or its other variants mRNA shown in any one.The gi number of the mRNA of each target gene is (" v " refers to and transcribe variant) shown in Table A.The polynucleotide sequence of said target mrna sequence or the target gene with mRNA sequence refer to the mRNA sequence shown in SEQ ID NO:1,2,10,7 or 11 or its any homologous sequence, and described homologous sequence preferably has and any one at least 70% the identity of the mRNA shown in SEQ ID NO:1,2,10,7 or 11, more preferably 80% identity, 90% or 95% identity even more preferably.Therefore, there is as described herein falling within the scope of the invention derived from SEQ ID NO:1,2,10,7 or 11 any one polynucleotide sequences of sudden change, change or modification.Term " mRNA polynucleotide sequence ", " mRNA sequence " and " mRNA " are used interchangeably.

As used herein, term " polynucleotide " and " nucleic acid " are used interchangeably, and refer to comprise the nucleotide sequence of thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA).Should be appreciated that these terms comprise any one analogue of the RNA that is made up of nucleotide analog or DNA equally.In the application's full text, mRNA sequence is shown and represents its corresponding gene.

" oligonucleotide " or " oligomer " refers to deoxyribonucleotide or the ribonucleoside acid sequence of approximately 2 to approximately 50 Nucleotide.Each DNA or RNA Nucleotide can be natural or synthetic independently, and/or modification or unmodified.Modification comprises the bonding between sugar moieties, base portion and/or the Nucleotide changing in oligonucleotide.Contain the molecule that comprises the deoxyribonucleotide of deoxyribonucleotide, ribonucleotide, modification, the ribonucleotide of modification, unconventional part and combination thereof according to the compound of many aspects of the present disclosure and embodiment.

" substantially complementary " refers to the complementarity that is greater than approximately 84% with another sequence.For example, in the duplex region being made up of 19 base pairs, mispairing produces 94.7% complementarity, and two mispairing produce approximately 89.5% complementarity, and 3 mispairing produce approximately 84.2% complementarity, thereby makes duplex region substantially complementary.Therefore, the substantially the same identity that is greater than approximately 84% with another sequence that refers to.

" Nucleotide " is intended to comprise can be natural or synthetic and/or deoxyribonucleotide and ribonucleotide that modify or unmodified.Modification comprises the bonding between sugar moieties, base portion and/or the ribonucleotide changing in oligoribonucleotide.As used herein, that term " ribonucleotide " is contained is natural and synthetic, unmodified with the ribonucleotide of modifying.Modification comprises the bonding between sugar moieties, base portion and/or the ribonucleotide changing in oligonucleotide.

Nucleotide can be selected from naturally occurring or synthetic modified base.Naturally occurring base comprises VITAMIN B4, guanine, cytosine(Cyt), thymine and uracil.The modified base of Nucleotide comprises inosine, xanthine, xanthoglobulin, 2-aminoadenine, 6-methyl, 2-propyl group and other alkyl VITAMIN B4, 5-halogen uridylic, 5-halogen cytosine(Cyt), 6-azepine cytosine(Cyt) and 6-azathymine, pseudouracil, 4-thiouracil, 8-halogen VITAMIN B4, 8-aminoadenine, 8-mercaptan VITAMIN B4, 8-mercaptan alkyl VITAMIN B4, 8-hydroxyadenine and other 8-substituted adenines, 8-halogen guanine, the amino guanine of 8-, 8-mercaptan guanine, 8-alkylthio guanine, 8-hydroxyl guanine and other 9 substituted guanines, other azepines and denitrification VITAMIN B4, other azepines and denitrification guanine, 5-trifluoromethyl uracil and 5-tri-flucytosines.In some embodiments, the one or more Nucleotide in oligomer are replaced by inosine.

According to some embodiments, the disclosure provides and comprises purposes unmodified and the inhibition oligonucleotide compound Nucleotide of modifying and/or unconventional part.This compound comprises the Nucleotide that is selected from least one modification that sugar-modified, base modification and internucleotide linkage modify, and can comprise the Nucleotide of DNA and modification, such as LNA (lock nucleic acid), ENA (nucleic acid of ethylidene bridge joint), PNA (peptide nucleic acid(PNA)), Arabinoside, phosphono carboxylic acid or phosphinocarboxylic acid's Nucleotide (PACE Nucleotide), mirror nuclei thuja acid or there is the Nucleotide of 6 carbon sugar.

All analogues of Nucleotide/oligonucleotide or modification all can be used for embodiment of the present invention, and prerequisite is that described analogue or modification can not produce significant disadvantageous effect to the function of Nucleotide/oligonucleotide.Acceptable modification comprises modification in modification, the internucleotide linkage of modification, base portion of sugar moieties and their combination.

Modification in the sugar-modified 2 ' part that comprises saccharide residue, and contain amino, fluorine, alkoxyl group (for example methoxyl group), alkyl, amino, fluorine, chlorine, bromine, CN, CF, imidazoles, carboxylate radical, sulfo-acid group (thioate), C ₁to C ₁₀low alkyl group, alkaryl or the aralkyl of low alkyl group, replacement, OCF ₃, OCN, O-, S-or N-alkyl; O-, S or N-thiazolinyl; SOCH ₃; SO ₂cH ₃; ONO ₂; NO ₂, N ₃; Heterocyclylalkyl; Heterocycle alkaryl; Aminoalkyl group amino; Gather the silica-based of alkylamino or replacement, etc., described in european patent number EP 0 586 520B1 or EP 0 618 925B1.

In one embodiment, the dsRNA molecule that can be used for method provided in this article, composition, combination, commercial package and test kit comprises at least one ribonucleotide that contains and 2 ' modify (" 2 ' is sugar-modified) on sugar moieties.In certain embodiments, it is sugar-modified that compound comprises 2 ' O-alkyl or 2 '-fluoro or 2 ' O-allyl group or any other 2 ', is optionally positioned in alternate position.It is also possible (for example, end modified) that other stabilizations are modified.In some embodiments, preferred 2 ' O-alkyl is that 2 ' O-methyl (methoxyl group) is sugar-modified.

In some embodiments, the main chain of oligonucleotide is modified and is comprised phosphoric acid-D-ribose entity, but also can comprise thiophosphoric acid-D-ribose entity, three esters, thioic acid sulfoacid, 2 '-5 ' bridge joint main chain (also can be described as 5 '-2 '), PACE etc.

As used herein, term " nucleotide analog of non-matching " refers to the nucleotide analog that comprises non-base pairing part, and these parts include but not limited to: 6 deaminize adenosine (nebularine), 4-Me-indoles, 3-nitro-pyrrole, 5-nitroindoline, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC.In some embodiments, non-base pairing nucleotide analog is ribonucleotide.In other embodiments, it is deoxyribonucleotide.In addition, prepare the analogue of polynucleotide, the structure of wherein one or more Nucleotide fundamentally changes and is more suitable for as treatment or experiment reagent.The example of nucleotide analog is peptide nucleic acid(PNA) (PNA), and wherein ribodesose (or ribose) phosphate backbone in DNA (or RNA) is substituted to the similar polyamide skeleton being present in peptide.Show, the resistance to enzyme liberating of PNA analogue, and have in longer body and vitro stability.Other modifications that can carry out oligonucleotide (for example comprise main polymer chain, ring-type main chain, acyclic main chain, thiophosphoric acid-D-ribose main chain, three ester main chains, thioic acid sulfoacid main chain, 2 '-5 ' bridge joint main chain, artificial nucleic acid, morpholino nucleic acid, glycol nucleic acid (GNA), threose nucleic acid (TNA), Arabinoside and mirror nuclei thuja acid, β-L-dezyribonucleoside, instead of β-D-dezyribonucleoside).The example of the dsRNA molecule that comprises LNA Nucleotide is at the people such as Elmen (NAR 2005,33 (1): have disclosed 439-447).The compound that can use according to the disclosure can use one or more to be inverted Nucleotide and for example be inverted thymidine or be inverted VITAMIN B4 synthetic (referring to the people such as such as Takei, 2002, JBC277 (26): 23800-06).

Other modifications are included in 5 ' and/or 3 ' end modified in part of oligonucleotide and are also referred to as end-blocking part.This type of end modified Nucleotide, lipid, peptide and inverted dealkalize base section that is selected from Nucleotide, modification.

" moieties or derivatives thereof " refers to straight or branched carbon part and itself contains or further contain the part of the functional group that comprises alcohol, phosphodiester, thiophosphatephosphorothioate, phosphoryl acetic ester, and comprise amine, carboxylic acid, ester, acid amides and aldehyde." hydrocarbon part " and " moieties " are used interchangeably.

" functional end-group " comprises halogen, alcohol, amine, carboxyl, ester, acid amides, aldehyde, ketone, ether.

The invention provides method, composition and combination for suppressing target gene expression in vivo.In certain embodiments, the method comprises being enough to using especially composition or the combination of siRNA (being siRNA) of oligoribonucleotide, the mRNA that described oligoribonucleotide target is transcribed by target gene by for example amount of RNA interference mechanism downward expression of target gene.Specifically, the expression that subject methods can be used for suppressing target gene is with treatment disease.Provide for target gene disclosed herein herein and can be used as therapeutical agent to treat composition and the combination of dsRNA molecule of multiple ear and vestibular system pathology.

Be provided for suppressing method, combination and the composition of hearing loss genes involved expression in vivo herein.In general, the amount that the method comprises being enough to lowering expression of target gene by for example RNA interference mechanism is used the especially combination of double-stranded RNA (for example siRNA) or the pharmaceutical composition that comprises described combination of oligoribonucleotide, described oligoribonucleotide said target mrna.Specifically, the expression that subject methods can be used for suppressing hearing loss genes involved is to treat disease disclosed herein or obstacle or illness.

Be provided for suppressing method, combination and the composition of HES1, HES5 and HEY2 expression in vivo herein.Be provided for suppressing the method and composition of HEY2, CDKN1B and NOTCH1 expression in vivo herein.In general, the method comprises being enough to using oligoribonucleotide by for example amount of RNA interference mechanism downward expression of target gene, especially double-stranded RNA (being dsRNA) or can produce the nucleic acid substances of dsRNA in cell, their targets are by the mRNA of HES1, HES5 and HEY2 gene or HEY2, CDKN1B and NOTCH1 genetic transcription.

dsRNA and RNA disturb

It is that one relates to phenomenon reticent after two strands (ds) RNA dependent gene specific transcriptional that RNA disturbs (RNAi).The initial effort of studying this phenomenon and handle experimentally mammalian cell is because of the failure of active, non-specific antiviral defense mechanism, this mechanism long dsRNA molecule of response and activate people such as (, Apoptosis, 2000.5:107-114) Gil.Afterwards, someone finds the gene specific RNAi in the double-stranded physical efficiency mediate mammalian of the RNA of 21 Nucleotide that synthesize cell, and do not stimulate the general antiviral defense mechanism (people such as Elbashir, Nature 2001, the people such as 411:494-498 and Caplen, PNAS 2001,98:9742-9747).Therefore, for the siRNA (siRNA) of short double-stranded RNA has been widely used for inhibition of gene expression and understand gene function.

RNA disturbs (RNAi) by siRNA (siRNA) people such as (, Nature 1998,391:806) Fire or microRNA (miRNA) mediation (Ambros V.Nature 2004,431:350-355; With Bartel DP.Cell.2004116 (2): 281-97).Corresponding process is commonly called gene silencing after specific transcriptional and in the time observing, is called as oppressive (quelling) in fungi while observation in plant.

SiRNA compound is to lower or the double-stranded RNA of reticent (suppressing wholly or in part) endogenous or foreign gene/mrna expression.RNA disturbs the ability that enters specific protein mixture based on some dsRNA material, then in described protein complex they be targeted to the degraded of complementary cell RNA specificity they.Therefore, the feature of rnai response is the restriction endonuclease complex body that comprises siRNA, is commonly called the silencing complex (RISC) of RNA induction, and the antisense strand of its mediation and siRNA duplex has the cracking of the single stranded RNA of complementary sequence.The cracking of target RNA can occur in the centre (people such as Elbashir, Genes Dev., 2001,15:188) with the region of the antisense strand complementation of siRNA duplex.More particularly, the dsRNA fragment (also referred to as short inhibitory RNA or " siRNA ") that longer dsRNA is digested to short (17-29bp) by III type RNA enzyme (DICER, DROSHA etc.) is (referring to people such as Bernstein, Nature, 2001, the people such as 409:363-6 and Lee, Nature, 2003,425:415-9).RISC protein complexes is identified these fragments and complementary mRNA.Whole process finishes (McManus and Sharp, Nature Rev Genet, 2002,3:737-47 with endonuclease to the cutting of said target mrna; Paddison and Hannon, Curr Opin Mol Ther.2003,5 (3): 217-24).(about the machine-processed details of these terms and proposition, referring to the people such as such as Bernstein, RNA.2001,7 (11): 1509-21; Nishikura, Cell.2001,107 (4): 415-8 and PCT publication number WO 01/36646).

Corresponding to selection and the synthetic existing wide coverage of the dsRNA compound of known; Referring to the people such as such as Ui-Tei, J Biomed Biotechnol.2006; 65052; The people such as Chalk, BBRC.2004,319 (1): 264-74; Sioud and Leirdal, Met.Mol Biol.; 2004,252:457-69; The people such as Levenkova, Bioinform.2004,20 (3): 430-2; The people such as Ui-Tei, NAR 2004,32 (3): 936-48.About the example that uses and prepare of siRNA of modifying, referring to people such as Braasch, Biochem., 2003,42 (26): 7967-75; The people such as Chiu, RNA, 2003,9 (9): 1034-48; PCT announces WO 2004/015107 (atugen); WO02/44321 (people such as Tuschl) and U.S. Patent number 5,898,031 and 6,107,094.

Multiple groups have described and have developed the carrier based on DNA that can generate siRNA in cell.The method is usually directed to transcribing of short hairpin RNA, and these RNA are effectively processed to form siRNA (people such as Paddison, PNAS USA 2002,99:1443-1448 in cell; The people such as Paddison, Genes & Dev 2002,16:948-958; The people such as Sui, PNAS USA 2002, the people such as 8:5515-5520 and Brummelkamp, Science 2002,296:550-553).These reports have described that generate can selectively targeted many endogenous and the method for the siRNA of exogenous expression's gene.

Research discloses, and siRNA can be effective in vivo in the Mammals that comprises the mankind.Specifically, the people such as Bitko have shown, can effectively treat mouse (Nat.Med.2005,11 (1): 50-55) for the specific siRNA of respiratory syncytial virus (RSV) nucleocapsid N gene when through intranasal administration.About the summary of the therapeutic application to siRNA, referring to for example Barik (Mol.Med 2005,83:764-773) and Chakraborty (Current Drug Targets20078 (3): 469-82).In addition, in human patients, carry out the clinical study (Kaiser, Am J Ophthalmol.2006142 (4): 660-8) with the short siRNA for the treatment of age-related macular degeneration (AMD) about target VEGFR1 acceptor.Be found in Durcan about siRNA as the other information of the purposes of therapeutical agent, 2008.Mol.Pharma.5 (4): 559 – 566; Kim and Rossi, 2008.BioTechniques 44:613-616; Grimm and Kay, 2007, JCI, 117 (12): 3633-41.

The dsRNA that can be used for combination treatment or composition is duplex oligoribonucleotide, therein, sense strand substantially with the fragment complementation of 18-40 continuous nucleotide of the mRNA polynucleotide sequence of target gene, and antisense strand substantially with sense strand complementation.In general, can tolerate and not reduce siRNA activity (referring to the people such as such as Czauderna, Nuc.Acids Res.2003,31 (11): 2705-2716) with some deviation of said target mrna sequence.SiRNA of the present invention destroy or do not destroy mRNA in the situation that on post-transcriptional level inhibition of gene expression.Not being bound by theory, can target mRNA there is specificity cracking and degraded and/or can suppress the translation of the information of institute's target in siRNA.

In some embodiments, dsRNA is blunt end at one or two end.More particularly, dsRNA can the 3'-of the 5'-end by the first chain and the second chain hold on the end limiting or by the 3'-of the first chain hold and the 5'-of the second chain to hold on the end limiting be blunt end.

In other embodiments, at least one of two chains can have overhanging of at least one Nucleotide at 5'-end, and this is overhang and can be made up of at least one deoxyribonucleotide.Article two, at least one of chain can also optionally have overhanging of at least one Nucleotide at 3'-end.This is overhang and can be made up of approximately 1 to approximately 5 Nucleotide.

The length of RNA duplex is approximately 18 to approximately 49 ribonucleotides, preferably 19 to 23 ribonucleotides.In addition, every chain (oligomer) can have independently and is selected from following length: approximately 18 to approximately 49 bases, preferably 18 to 23 bases, more preferably 19,20 or 21 ribonucleotides.

In addition, in certain preferred aspects, perfect complementary between described the first chain and target nucleic acid.In some embodiments, described chain is substantially complementary,, has one, two or maximum three mispairing between described the first chain and target nucleic acid that is.

In addition, the 5'-of siRNA the first chain end can be connected to the 3'-end of the second chain, or the 3'-of the first chain end can be connected to the 5'-end of the second chain, and described bonding is conventionally to have the preferably nucleic acid joint of approximately 3 to approximately 10 length of nucleotides of 3-100 Nucleotide.

The dsRNA compound that can be used for method disclosed herein, combination and composition has structure and the modification of giving following one or more aspects: the immune response of the toxicity of the activity of increase, the stability of increase, reduction, miss the target effect and/or the reduction of reduction.SiRNA structure is applicable to can be used for the double-stranded RNA of method disclosed herein, combination and composition valuably as disclosed herein, for preventing or reducing expression of target gene, target gene especially discussed in this article.

According to an aspect, the disclosure provides the combination of double chain oligonucleotide or the purposes of composition of chemically modified, and this oligonucleotide comprises at least one and is selected from the modified nucleotide that sugar-modified, base modification and internucleotide linkage are modified.Therefore, the double chain oligonucleotide compound that can be used for the chemically modified of method provided in this article, composition and combination can comprise the Nucleotide of modification, such as DNA, LNA (lock nucleic acid), ENA (nucleic acid of ethylidene bridge joint), PNA (peptide nucleic acid(PNA)), PACE, mirror nuclei thuja acid or have the Nucleotide of 6 carbon sugar.The example of PACE Nucleotide and analogue has disclosed in the U.S. Patent number 6,693,187 and 7,067,641 being all incorporated herein by reference.Oligonucleotide can also comprise 2 ' O-methyl or 2 '-fluorine or 2 ' O-allyl group or any other 2 ' modification, optionally in alternate position.It is not obvious that to fall that SA other stabilizations modify be also possible (for example end modified).The main chain of the active part of oligonucleotide can comprise phosphoric acid-D ribose entity, but also can comprise the modification of thiophosphoric acid-D-ribose entity, three esters, thioic acid sulfoacid, 2 '-5 ' bridge main chain (also can be described as 5 '-2 '), PACE or any other type.5 ' and/or 3 ' end modified in part of oligonucleotide is also possible.This type of is end modified can be lipid, peptide, sugar, inverted dealkalize base section or other molecules.

the chemosynthesis of oligonucleotide compound

Oligonucleotide compound for method disclosed herein, composition, combination, commercial package and test kit can be synthetic by any method for the synthesis of ribose core (or ribodesose core) oligonucleotide well known in the art.This type of synthesis example is as at Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucage and Iyer, Tetrahedron 1993; The people such as 49:6123-6194 and Caruthers, Methods Enzymol.1987; In 154:287-313, describe to some extent; The synthesis example of thioic acid sulfoacid is as at Eckstein, Annu.Rev.Biochem.1985; In 54:367-402, describe to some extent; Synthesizing at Sproat of RNA molecule, Humana Press2005, Herdewijn P. edits; In Kap.2:17-31, describe to some extent, and corresponding downstream process is for example people such as Pingoud, IRL Press 1989, Oliver R.W.A. edits; In Kap.7:183-208, describe to some extent.

Other synthesis programs are known in the art, for example, as people such as Usman, 1987, J.Am.Chem.Soc., 109,7845; The people such as Scaringe, 1990, NAR., 18,5433; The people such as Wincott; 1995; NAR.23; the people such as 2677-2684 and Wincott, 1997, Methods Mol.Bio.; 74; program described in 59, and these programs can utilize common nuclease protection and coupling group, such as the phosphoramidite of dimethoxytrityl and the 3 '-end of 5 '-end.(for example 2 '-O-the is methylated) Nucleotide of modifying and the Nucleotide of unmodified mix as required.

The oligonucleotide that can use according to embodiment provided in this article can synthesize individually and be bonded together after synthetic, for example, by connection (people such as Moore, 1992, Science 256,9923; The people such as Draper, International Patent Publication No. W WO 93/23569; The people such as Shabarova, 1991, NAR 19,4247; The people such as Bellon, 1997, Nucleosides & Nucleotides, 16,951; The people such as Bellon, 1997, Bioconjugate Chem.8,204) or by synthetic and/or go to protect after hybridization.

It should be noted that and can use commercially available equipment (particularly can derive from Applied Biosystems); And prepare oligonucleotide according to sequence disclosed herein.Can use method well known in the art to connect overlapping chemosynthesis fragment to (for example, referring to U.S. Patent number 6,121,426).Separately synthetic each chain, then annealing each other in pipe.Then, by HPLC, the single stranded oligonucleotide of double-stranded siRNA and unannealed (for example,, because wherein a kind of excessive) is separated.About dsRNA or the dsRNA fragment that can use according to embodiment of the present disclosure, can synthesize two or more these type of sequences and link together, for method disclosed herein, composition and combination.

Compound used according to the invention also can be synthetic by series connection synthetic technology, as described in U.S. Patent Publication number 2004/0019001 (McSwiggen), wherein that two siRNA chains are all synthetic with following form: single continuous oligonucleotide fragment or joint (it is cleaved so that independent siRNA fragment to be provided subsequently) chain separating or the chain of hybridizing and allow purifying siRNA duplex by cleavable in for example.Joint can be polynucleotide joint or non-nucleotide joint.

The disclosure is provided for treating the pharmaceutical composition that comprises three kinds of dsRNA molecules of any disease as herein described and illness, thus, wherein at least two kinds of molecules can be in pharmaceutical composition with generate equate or the amount physical mixed of useful activity together, or bonding covalently or non-covalently, or be bonded together by the nucleic acid joint within the scope of the preferred 2-50 of 2-100 or 2-30 length of nucleotides.In one embodiment, dsRNA molecule passes through double-strandednucleic acid structure as described herein and forms, and wherein dsRNA molecule is selected from oligonucleotide as herein described.Therefore, dsRNA molecule covalently or non-covalently bonding or by joint engage to form series connection or triplet state dsRNA compound.Length of this type of series connection dsRNA molecule that comprises two siRNA sequences is generally 38-150 Nucleotide, and more preferably length is 38 or 40-60 Nucleotide, and connect correspondingly longer while comprising more than two siRNA sequences in molecule.Also imagine the long series connection compound of the longer sequence formation of the siRNA (for example long dsRNA) being produced by inner cell processing by two or more codings, as the series connection molecule of two or more shRNA that encode.This type of series connection molecule is also regarded as a part of this disclosure.Imagine the compound that comprises two (series connection) disclosed herein or more (RNAistar) dsRNA sequence.The example of this type of " series connection " or " star " molecule provides to some extent transferring in present assignee the PCT patent publication No. WO 2007/091269 that is incorporated herein by reference of entirety.

The dsRNA molecule of target HES1, HES5 and HEY2 or HEY2, CDKN1B and NOTCH1 can be the main active component in pharmaceutical composition.Suppressing described other gene may have and be added or synergy simultaneously in the time for the treatment of disease disclosed herein.

In addition, dsRNA disclosed herein or comprise any nucleic acid molecule of this type of dsRNA that encodes can connect or bonding (covalently or non-covalently) to the antibody (comprising aptamers molecule) of chemoattractant molecule in the cell surface for expressing on target cell, to realize the targeting strengthening to treat disease disclosed herein.For example, anti-Fas antibody (preferably neutralizing antibody) can be combined with any dsRNA (covalently or non-covalently).The aptamers that can play a role as part/antibody in another example, can be combined with any dsRNA (covalently or non-covalently).

Nucleic acid molecule disclosed herein can directly be sent or send by virus or non-virus carrier.In the time directly sending, conventionally making calling sequence is resistance to nuclease.Or, sequence can be mixed in expression cassette or construct, make calling sequence as mentioned below at cells.Conventionally, construct comprises suitable regulating and controlling sequence or promotor to allow sequence to express in target cell.Optionally for sending the commercially available acquisition of carrier of compound of the present invention, and can modify to send compound of the present invention by method known to those skilled in the art.

can be used for the dsRNA of combination treatment

In the particular of composition provided herein, combination, method, commercial package and test kit, double chain oligonucleotide (for example dsRNA) has with corresponding unmodified double chain oligonucleotide Compound Phase than increasing activity, increase stability, at utmost reduce toxicity and/or affect double chain oligonucleotide the modification that is delivered to middle ear or inner ear.Double chain oligonucleotide molecule is designed to lower expression of target gene and weakens target gene function.In certain embodiments, target gene is transcribed into any one of the mRNA polynucleotide shown in SEQ ID NO:1,2,7,10 and 11.At composition provided herein, combination, method, in the particular of commercial package and test kit, double chain oligonucleotide (for example dsRNA) has and is selected from the SEQ ID NO:23-1495 or the 26667-26706 (HES1) that can be used for generating chemically modified double chain oligonucleotide molecule, SEQ ID NO:1496-2703 or 26707-26732 (HES5), SEQ ID NO:13004-16621 or 26779-26788 (HEY2), sense strand sequence and the antisense strand sequence of the sense strand oligonucleotide shown in SEQ ID NO:7444-10533 or 26867-26900 (CDKN1B) or SEQ ID NO:16622-26666 or 26901-26912 (NOTCH1) and corresponding antisense strand oligonucleotide.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, for example, in each of double chain oligonucleotide molecule (dsRNA molecule), antisense strand can be 18 to 49 Nucleotide long (for example 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48 or 49 Nucleotide are long); Or 18-35 Nucleotide is long; Or 18-30 Nucleotide is long; Or 18-25 Nucleotide is long; Or 18-23 Nucleotide is long; Or 19-21 Nucleotide is long; Or 25-30 Nucleotide is long; Or 26-28 Nucleotide is long.In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, for example, in each of double chain oligonucleotide molecule (dsRNA molecule), antisense strand is that 19 Nucleotide are long.Similarly, the sense strand of double chain oligonucleotide molecule (for example dsRNA molecule) can be 18 to 49 Nucleotide long (for example 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48 or 49 Nucleotide are long); Or 18-35 Nucleotide is long; Or 18-30 Nucleotide is long; Or 18-25 Nucleotide is long; Or 18-23 Nucleotide is long; Or 19-21 Nucleotide is long; Or 25-30 Nucleotide is long; Or 26-28 Nucleotide is long.In the multiple preferred embodiment of composition, combination, method, commercial package and test kit as disclosed herein, for example, in each double chain oligonucleotide (dsRNA molecule), sense strand is that 19 Nucleotide length and antisense strand are that 19 Nucleotide are long.In the multiple preferred embodiment of composition, combination, method, commercial package and test kit as disclosed herein, the duplex region of double chain oligonucleotide molecule (for example dsRNA molecule) can be 18-49 Nucleotide long (for example approximately 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48 or 49 Nucleotide is long); 18-35 Nucleotide is long; Or 18-30 Nucleotide is long; Or 18-25 Nucleotide is long; Or 18-23 Nucleotide is long; Or 18-21 Nucleotide is long; Or 25-30 Nucleotide is long; Or 25-28 Nucleotide is long.In the multiple preferred embodiment of composition, combination, method, commercial package and test kit as disclosed herein, the duplex region of double chain oligonucleotide molecule is that 19 Nucleotide are long.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, sense strand and the antisense strand of double chain oligonucleotide (for example dsRNA molecule) are independent oligonucleotide chain.In some embodiments, independent sense strand and antisense strand for example, form duplex structure via hydrogen bond (Watson-Crick base pairing), also referred to as duplex.In some embodiments, one or more nucleotide pairs form non-Watson-Crick base-pair.In some embodiments, sense strand and antisense strand are two covalently bound each other independent chains.In other embodiments, sense strand and antisense strand are parts with the single core oligonucleotide in sense and antisense region; In some preferred embodiments, oligonucleotide has hairpin structure.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide is with respect to the symmetry of overhanging, and has blunt end on two ends.In other embodiments, double chain oligonucleotide is dsRNA molecule, and it is with respect to the symmetry of overhanging, and on two ends of dsRNA molecule, has the combination that Nucleotide or non-nucleotide or Nucleotide and non-nucleotide are overhang.In certain preferred aspects, nucleic acid molecule is dsRNA molecule, and it is with respect to the symmetry of overhanging, and has on blunt end and another end at molecule and have and overhang on an end of molecule.In some embodiments, asymmetric dsRNA molecule has 3 ' in a side of the duplex being present on sense strand-overhang; And be present in the blunt end on the opposite side of the molecule on 5 '-end of 5 ' of sense strand-end and antisense strand.In some embodiments, asymmetric dsRNA molecule has 5 ' in a side of the duplex being present on sense strand-overhang; And be present in the blunt end on the opposite side of the molecule on 3 '-end of 3 ' of sense strand-end and antisense strand.In other embodiments, asymmetric dsRNA molecule has 3 ' in a side of the duplex being present on antisense strand-overhang; And be present in the blunt end on the opposite side of the molecule on 5 '-end of 5 ' of sense strand-end and antisense strand.In some embodiments, asymmetric dsRNA molecule has 5 ' in a side of the duplex being present on antisense strand-overhang; And be present in the blunt end on the opposite side of the molecule on 3 '-end of 3 ' of sense strand-end and antisense strand.In some embodiments, overhang and overhang for Nucleotide, in other embodiments, overhang and overhang for non-nucleotide.In some embodiments, overhanging is 5 ' to overhang; In alternate embodiment, overhanging is 3 ' to overhang.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide has hairpin structure (having sense strand and antisense strand on an oligonucleotide), wherein on an end, there is ring texture, and there is blunt end on another end.In some embodiments, double chain oligonucleotide has hairpin structure, wherein on an end, has ring texture, overhangs and have on another end; In certain embodiments, overhang be 3 '-overhang; In certain embodiments, overhang be 5 '-overhang; In certain embodiments, overhang and be positioned on sense strand; In certain embodiments, overhang and be positioned on antisense strand.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can comprise one or more Nucleotide of modifying or modifying as described herein.For example, double chain oligonucleotide (for example dsRNA molecule) can comprise have modification sugared modified nucleotide, there is the modified nucleotide of the core base of modification, or there is the modified nucleotide of phosphate group, the phosphodiester backbone of modification and/or the terminal phosphate group of modification of modification.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can have the ribonucleotide of one or more sugar moieties that comprise modification (for example, as described herein).The limiting examples of the sugar moieties of modifying is the sugar moieties that 2 ' alkoxyl group is modified.In some preferred embodiments, nucleic acid comprises the sugar-modified ribonucleotide of at least one 2 '-O-methyl.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can have one or more core bases of for example modifying as described herein.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can have one or more for example modifications to phosphodiester backbone as described herein.

In some embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can have one or more phosphate groups of for example modifying as described herein.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can comprise the antisense strand of unmodified and have the sense strand of one or more modifications.In some embodiments, double chain oligonucleotide (for example dsRNA molecule) can comprise the sense strand and the antisense strand with one or more modifications of unmodified.In preferred embodiments, the Nucleotide that double chain oligonucleotide (for example dsRNA molecule) can comprise one or more modifications in sense strand and antisense strand.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can comprise phosphate group (, 5 '-terminal phosphate group) at 5 ' end of sense strand and/or antisense strand.In some embodiments, double chain oligonucleotide can comprise phosphate group at 5 ' end of antisense strand.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can comprise phosphate group (, 3 '-terminal phosphate group) at 3 ' end of sense strand and/or antisense strand.In some embodiments, double chain oligonucleotide can comprise phosphate group at 3 ' end of antisense strand.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide (for example dsRNA molecule) can comprise phosphate group at 3 ' end of antisense strand and sense strand.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, for example, for double chain oligonucleotide (dsRNA molecule), the antisense strand of nucleic acid molecule and sense strand are in all phosphorylations not of 3 ' end and 5 ' end.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A1) independently:

(A1) 5 ' (N) x – Z, 3 ' (antisense strand)

3 ' Z '-(N ') y – z " 5 ' (sense strand)

Wherein each N and N ' are can unmodified or adorned ribonucleotide, or unconventional part; Wherein each of (N) x and (N ') y is oligonucleotide, and wherein each continuous N or N ' join next N or N ' to by covalent linkage;

Wherein each of Z and Z ' has an independent existence or does not exist, if but while existence, comprise independently 1-5 continuous nucleotide, 1-5 continuous non-nucleotide part or its combination of the covalently bound 3 ' end at its existing chain;

Wherein z " can exist or not exist, if but while existing, be the end-blocking part of the covalently bound 5 ' end at (N ') y;

Each of x and y is 18 to 40 integer independently;

The wherein sequence of (N ') y and (N) the sequence complementation of x; And the mRNA of mRNA, coding CDKN1B of mRNA, coding HEY2 that wherein (N) x comprises the mRNA, the coding HES5 that are selected from coding HES1 and the antisense sequences of the mRNA of the mRNA of coding NOTCH1.

At composition as disclosed herein, combination, method, in the multiple embodiments of commercial package and test kit, at least one double chain oligonucleotide has structure (A1) independently, (N) x comprises antisense sequences and (N ') y includes adopted sequence, as SEQ ID NO:23-693 and 26691-26706 (HES1), SEQ ID NO:1496-2029 and 26725-26732 (HES5), SEQ ID NO:7444-9007 and 26887-26900 (CDKN1B), SEQ ID NO:13004-14801 and 26785-26788 (HEY2), SEQ ID NO:16622-18643 and 26922 – 26912 (NOTCH1) are shown in any one.In some embodiments, preferred (N) x and (N ') y are if SEQ ID NO:26691-26706 (HES1), SEQ ID NO:26725-26732 (HES5), SEQ ID NO:26887-26900 (CDKN1B), SEQ ID NO:26785-26788 (HEY2), SEQ ID NO:26922 – 26912 (NOTCH1) are as shown in any one.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, the covalent linkage that at least one double chain oligonucleotide has independently structure (A1) and connects each N continuous and/or N ' is phosphodiester bond.

In the multiple embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide there is independently structure (A1) x=y and x and y each be 19,20,21,22 or 23.In preferred embodiments, x=y=19.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide comprises DNA part or the mispairing at the 1st (5 ' end) Yu the target of antisense strand.In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure as follows (A2):

(A2) 5 ' N1-(N) x-Z 3 ' (antisense strand)

3 ' Z '-N2-(N ') y – z " 5 ' (sense strand)

Wherein each N1, N2, N and N ' are Nucleotide unmodified or that modify independently, or unconventional part;

Wherein each of (N) x and (N ') y is each continuous N wherein or N ' and joins to by covalent linkage the oligonucleotide of adjacent N or N ';

Wherein each of x and y is the integer between 17 and 39 independently;

Wherein N2 is covalently bound to (N ') y;

Wherein N1 is covalently bound to (N) x and with said target mrna mispairing or be the complementary DNA part of said target mrna;

Wherein N1 is the part of the choosing group that freely uridine, deoxyuridine, ribose thymidine, ribodesose thymidine, adenosine or Desoxyadenosine, dealkalize base ribose part and dealkalize base ribodesose part natural or that modify form;

Wherein z " can exist or not exist, if but while existing, be the end-blocking part of the covalently bound 5 ' end at N2-(N ') y;

Wherein each of Z and Z ' has an independent existence or does not exist, if but while existence, be 1-5 continuous nucleotide, the individual continuous non-nucleotide part of 1-5 or its combination of the covalently bound 3 ' end at its existing chain independently; And

Wherein the sequence of (N ') y has complementarity with (N) sequence of x; And wherein the sequence of (N) x is for being selected from the antisense sequences of the continuous sequence in HES1mRNA (SEQ ID NO:1), HES5mRNA (SEQ ID NO:2), HEY2mRNA (SEQ ID NO:10), CDKN1B mRNA (SEQ ID NO:7) or NOTCH1mRNA (SEQ ID NO:11).

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), wherein the sequence of (N ') y and (N) the sequence complementation of x; And wherein the sequence of (N) x comprises antisense sequences and (N ') y includes adopted sequence, if SEQ ID NO:694-1495 and 26667-26690 (HES1), SEQ ID NO:2030-2703 and 26707-26724 (HES5), SEQ ID NO:9008-10533 and 26867-26886 (CDKN1B), SEQ ID NO:14802-16389 and 26779-26784 (HEY2), SEQ ID NO:18644-26666 and 26901-26910 (NOTCH1) are as shown in any one.Preferably (N) x and (N ') y are if SEQ ID NO:26667-26690 (HES1), SEQ ID NO:26707-26724 (HES5), SEQ ID NO:26867-26886 (CDKN1B), SEQ ID NO:26779-26784 (HEY2), SEQ ID NO:26901-26910 (NOTCH1) are as shown in any one.The molecule of being contained by the description of structure (A2) is herein also referred to as " 18+1 " or " 18+1 aggressiveness ".In some embodiments, can be used for generating N2-(the N ') y of the double chain oligonucleotide with structure (A2) and N1-(N) x shown in Table I-V, refer in particular to the sequence that is decided to be " 18+1 " type.In certain preferred aspects, (N) x and (N ') y are selected from the sequence pair shown in Table I-V.

In the certain preferred embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide occupy structure (A2), the sequence of (N ') y and (N) the sequence complete complementary of x.In multiple embodiments, and N2-(N ') sequence of y and the sequence complementation of N1-(N) x.In some embodiments, (N) x comprise with the said target mrna of SEQ ID NO:1,2,10,7 or 11 shown in any one in the antisense sequences of approximately 17 to approximately 39 continuous nucleotide complete complementaries.In other embodiments, (N) x comprise with the said target mrna of SEQ ID NO:1,2,10,7 or 11 shown in any one in approximately 17 to approximately 39 continuous nucleotides complementary antisense sequences substantially.In some embodiments, at least one double chain oligonucleotide has structure (A2) and N1 and N2 formation Wo Sen-Ke Like base pair.In other embodiments, at least one double chain oligonucleotide has structure (A2) and N1 and N2 formation Fei Wosen-Ke Like base pair.In some embodiments, between ribonucleotide and deoxyribonucleotide, form base pair.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), x=y=18, x=y=19 or x=y=20.In preferred embodiments, x=y=18.In the time of x=18 in N1-(N) x, N1 refers to the 1st, and 2-19 position is included in (N) ₁₈in.In the time of y=18 in N2-(N ') y, N2 refers to the 19th, and 1-18 position is included in (N ') ₁₈in.In some embodiments, at least one double chain oligonucleotide has structure (A2), N1 be covalently bound to (N) x and with the said target mrna mispairing shown in SEQ ID NO:1,2,10,7 or 11.In multiple embodiments, at least one double chain oligonucleotide has structure (A2), N1 be covalently bound to (N) x and for the DNA part of the said target mrna complementation shown in SEQ ID NO:1,2,10,7 or 11.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), and the N1 that the uridine that antisense strand is the 1st is selected from natural or adenosine, Desoxyadenosine, uridine, deoxyuridine (dU), ribose thymidine or deoxythymidine that modify replaces.In multiple embodiments, at least one double chain oligonucleotide has structure (A2), and N1 is selected from adenosine, Desoxyadenosine or deoxyuridine natural or that modify.For example, in some embodiments, the cytidine of the 1st is substituted by VITAMIN B4 or uridine; The guanosine of the 1st is substituted by VITAMIN B4 or uridine; Or VITAMIN B4 is substituted by uridine.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), and the guanosine of antisense strand the 1st (N1) is replaced by adenosine, Desoxyadenosine, uridine, deoxyuridine, ribose thymidine or deoxythymidine natural or that modify.In multiple embodiments, N1 is selected from adenosine, Desoxyadenosine, uridine or deoxyuridine natural or that modify.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), and the cytidine of antisense strand the 1st (N1) is replaced by adenosine, Desoxyadenosine, uridine, deoxyuridine, ribose thymidine or deoxythymidine natural or that modify.In multiple embodiments, N1 is selected from adenosine, Desoxyadenosine, uridine or deoxyuridine natural or that modify.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), and the adenosine of antisense strand the 1st (N1) is replaced by Desoxyadenosine, deoxyuridine, ribose thymidine or deoxythymidine natural or that modify.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2), and N1 and N2 form base pair between uridine natural or that modify or deoxyuridine and adenosine or Desoxyadenosine.In other embodiments, N1 and N2 form base pair between deoxyuridine natural or that modify and adenosine.

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide molecule is also referred to as " duplex ".In some embodiments, at least one double chain oligonucleotide has structure (A2), and the double chain oligonucleotide dsRNA that is chemical modification.

In some preferred embodiment of composition, combination, method, commercial package and test kit as disclosed herein, double chain oligonucleotide molecule has structure (A2), and x=y=18.In some embodiments, x=y=18, and (N) x is made up of the antisense oligonucleotide being present in SEQ ID NO:694-1495 and 26667-26690 (HES1), SEQ ID NO:2030-2703 and 26707-26724 (HES5), SEQ ID NO:9008-10533 and 26867-26886 (CDKN1B), SEQ ID NO:14802-16389 and 26779-26784 (HEY2), SEQ ID NO:18644-26666 and 26901-26910 (NOTCH1).

In some embodiments of composition, combination, method, commercial package and test kit as disclosed herein, at least one double chain oligonucleotide has structure (A2) and N1 and is selected from the uridine of natural uridine and modification.In some embodiments, N1 is natural uridine.In some embodiments, (N) x comprises antisense oligonucleotide and (N ') y comprises the MODN that has that is present in the sequence centering shown in SEQ ID NO:694-1495 (HES1), SEQ ID NO:2030-2703 (HES5), SEQ ID NO:9008-10533 (CDKN1B), SEQ ID NO:14802-16389 (HEY2), SEQ ID NO:18644-26666 (NOTCH1).

At composition as disclosed herein, combination, method, in some embodiments of commercial package and test kit, at least one double chain oligonucleotide has structure (A2), x=y=18 and N1-(N) x comprises antisense oligonucleotide and N2-(N ') y comprises and is present in SEQ ID 26667-26690 (HES1), SEQ ID NO:26707-26724 (HES5), SEQ ID NO:26867-26886 (CDKN1B), SEQ ID NO:26779-26784 (HEY2), sequence centering shown in SEQ ID NO:26901-26910 (NOTCH1) have a MODN.

In some embodiments, at least one double chain oligonucleotide has structure (A2), x=y=18 and N1 be selected from natural or modify uridine, natural or modify VITAMIN B4 and natural or modify thymidine.

In some embodiments, at least one double chain oligonucleotide has structure (A2), and wherein N1 is the sugar-modified uridine of 2 ' OMe or the sugar-modified adenosine of 2 ' OMe.In certain embodiments, at least one double chain oligonucleotide has structure (A2), and N2 is the sugar-modified ribonucleotide of 2 ' OMe or deoxyribonucleotide.

In some embodiments of structure (A1) and/or structure (A2), each N is made up of the ribonucleotide of unmodified.In some embodiments of structure (A1) and/or structure (A2), each N ' is made up of the ribonucleotide of unmodified.In preferred embodiments, deoxyribonucleotide or the unconventional part of the deoxyribonucleotide of at least one ribonucleotide that comprises chemically modified of N and/or N ', unmodified, chemically modified.In some embodiments, this unconventional part is selected from mirror nuclei thuja acid, dealkalize base ribose part and dealkalize base ribodesose part.In some embodiments, this unconventional part is mirror nuclei thuja acid, preferably L-DNA part.In some embodiments, at least one of N or N ' comprises the sugar-modified ribonucleotide of 2 ' OMe.

In some embodiments of structure (A1) and/or structure (A2), the sequence of (N ') y and (N) the sequence complete complementary of x.In other embodiments of structure (A1) and/or structure (A2), the sequence of (N ') y is substantially complementary with the sequence of (N) x.

In some embodiments of structure (A1) and/or structure (A2), (N) x comprise with the said target mrna of SEQ ID NO:1,2,10,7 or 11 shown in any one in the antisense sequences of approximately 17 to approximately 39 continuous nucleotide complete complementaries.In other embodiments of structure A1 and/or structure A2, (N) x comprise with the said target mrna of SEQ ID NO:1,2,10,7 or 11 shown in any one in approximately 17 to approximately 39 continuous nucleotides complementary antisense sequences substantially.In some embodiments of structure (A1) and/or structure (A2), dsRNA compound is blunt end, for example, wherein z ", Z and Z ' each do not exist.In alternative embodiment, z ", at least one existence of Z or Z '.

In multiple embodiments, Z and Z ' comprise Nucleotide one or more covalently bound modifications and/or unmodified independently, comprise deoxyribonucleotide and ribonucleotide, or one or more unconventional parts, for example inverted dealkalize base ribodesose part or dealkalize base ribose part or mirror nuclei thuja acid; One or more non-nucleotide C3 part or derivatives thereofs, non-nucleotide C4 part or derivatives thereof or non-nucleotide C5 part or derivatives thereof, non-nucleotide amino-C6 part or derivatives thereof, as defined herein, etc.In some embodiments, Z ' does not exist and Z exists and comprise one or more non-nucleotide C3 parts.In some embodiments, Z does not exist and Z ' exists and comprise one or more non-nucleotide C3 parts.In some embodiments, each of Z and Z ' comprises one or more non-nucleotide C3 parts or one or more non-nucleotide amino-C6 part independently.In some embodiments, z " there is and be selected from mirror nuclei thuja acid, dealkalize base section and inverted dealkalize base section.In structure (A1) and/or some embodiments (A2), each of Z and Z' comprises dealkalize base section, for example ribodesose dealkalize base section (being called " dAb " herein) or ribose dealkalize base section (being called " rAb " herein).In some embodiments, each of Z and/or Z' comprises two covalently bound dealkalize base section and is for example dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb, and wherein each several part covalency is attached to adjacent part, preferably via the key based on phosphorus.In some embodiments, the key based on phosphorus comprises thiophosphatephosphorothioate, phosphonoacetic acid ester or phosphodiester bond.In preferred embodiments, the key based on phosphorus is phosphodiester bond.

In some embodiments, each of Z and/or Z' comprises moieties independently, optionally partly (C3) or derivatives thereof of propane [(CH2) 3], comprises the phosphorus derivant (" C3Pi ") of propyl alcohol (C3OH) and propylene glycol.In some embodiments, each of Z and/or Z' comprises two moieties, and is C3Pi-C3OH in some instances.In the example of C3Pi-C3OH, 3 ' end of antisense strand and/or 3 ' end of sense strand are attached to C3 part via the key covalency based on phosphorus, and C3 part is covalently bound to C3OH part via the key based on phosphorus.In some embodiments, the key based on phosphorus comprises thiophosphatephosphorothioate, phosphonoacetic acid ester or phosphodiester bond.In preferred embodiments, the key based on phosphorus is phosphodiester bond.

In structure (A1) and specific embodiments (A2), Z comprises C3Pi-C3OH.In structure (A1) and specific embodiments (A2), Z ' comprises C3Pi or C3OH.In structure (A1) and some embodiments (A2), double chain acid molecule comprises covalency and is attached to the C3Pi-C3OH part of 3 ' end of antisense strand, and covalency is attached to C3Pi or the C3OH part of 3 ' end of sense strand.

In some embodiments of structure (A1) and/or structure (A2), each N is made up of the ribonucleotide of unmodified.In some embodiments of structure (A1) and/or structure (A2), each N ' is made up of the ribonucleotide of unmodified.In preferred embodiments, at least one of N and/or N ' is for containing the ribonucleotide of chemically modified, the deoxyribonucleotide of unmodified, deoxyribonucleotide or the unconventional part of chemically modified.

In other embodiments, the compound of structure (A1) and/or structure (A2) comprises the ribonucleotide that at least one modifies in its saccharide residue.In some embodiments, this compound comprises modification 2 ' of saccharide residue.In some embodiments, the modification of 2 ' comprises existence amino, fluorine, alkoxyl group or moieties.In certain embodiments, 2 ' modification comprises alkoxyl group part.In preferred embodiments, alkoxyl group part is methoxyl group part (also referred to as 2 '-O-methyl, 2 ' OMe, 2 ' OMe, 2 '-OCH3).In some embodiments, nucleic acid compound comprises the sugar-modified alternately ribonucleotide of 2 ' OMe in the one or both of sense strand and antisense strand.In other embodiments, compound only comprises the sugar-modified ribonucleotide of 2 ' OMe in antisense strand (N) x or N1-(N) x.In some embodiments, the Nucleotide of the sugar-modified ribonucleotide of 2 ' OMe and unmodified alternately.In certain embodiments, for example ribonucleotide unmodified of the 10th in 19-aggressiveness chain of the intercalated nucleus sugar nucleotide of antisense strand.In multiple embodiments, nucleic acid compound comprises the sugar-modified ribonucleotide of at least 52 ' OMe that replace and the ribonucleotide of unmodified.In other embodiments, the ribonucleotide that structure (A1) and/or compound (A2) comprise modification in the position replacing, wherein modified in its saccharide residue at (N) x or the 5 ' end of N1-(N) x and each ribonucleotide of 3 ' end, and (N ') y or the 5 ' end of N2-(N) y and each ribonucleotide unmodified in its saccharide residue of 3 ' end.In multiple embodiments, the ribonucleotide in alternate position is modified in 2 ' position of saccharide residue.

In some embodiments, nucleic acid compound comprises the sugar-modified ribonucleotide of at least 52 ' OMe that replace and the ribonucleotide of unmodified, for example, at the 1st, 3,5,7 and 9 or at the 11st, 13,15,17,19 (5 ' >3 ').In some embodiments, N1-(N) x of (N) x of structure (A1) or structure (A2) comprises the sugar-modified ribonucleotide of 2 ' OMe at 2,4,6,8,11,13,15,17 and 19.In some embodiments, N1-(N) x of (N) x of structure (A1) or structure (A2) comprises the sugar-modified ribonucleotide of 2 ' OMe at the 1st, 3,5,7,9,11,13,15,17 and 19.In some embodiments, N1-(N) x of (N) x of structure (A1) or structure (A2) comprises the sugar-modified ribonucleotide of 2 ' OMe in one or more pyrimidines.

In structure (A1) and/or some embodiments (A2), sense strand and antisense strand are at 3 ' end and in all phosphorylations not of 5 ' end.In other embodiments, the one or both of sense strand and/or antisense strand is at 3 ' terminal phosphate.In other embodiments, the one or both of sense strand and/or antisense strand is at 5 ' terminal phosphate.

In some embodiments, duplex molecule disclosed herein comprises one or more in following modification:

N at least one of from 5 ' end of antisense strand the 5th, 6,7,8 or 9 is selected from DNA, TNA, 2 ' 5 ' Nucleotide or mirror nuclei thuja acid;

N ' the 9th or at least one of 10 from 5 ' end of sense strand is selected from TNA, 2 ' 5 ' Nucleotide and pseudouridine;

N ' in 4,5 or 6 continuous positions of the 3 ' end of (N ') y comprises 2 ' 5 ' ribonucleotides;

It is 2 ' sugar-modified that one or more pyrimidine ribonucleotides exist in sense strand, antisense strand or sense strand and antisense strand.

In some embodiments, the combination that duplex molecule disclosed herein comprises following modification:

Antisense strand comprises DNA, TNA, 2 ' 5 ' Nucleotide or mirror nuclei thuja acid at least one of the 5th, 6,7,8 or 9 from 5 ' end;

Sense strand from 5 ' end the 9th or 10 comprise at least one in TNA, 2 ' 5 ' Nucleotide and pseudouridine; And

One or more pyrimidine ribonucleotides exist 2 ' to modify in sense strand, antisense strand or sense strand and antisense strand.

Antisense strand comprises DNA, 2 ' 5 ' Nucleotide or mirror nuclei thuja acid at least one of the 5th, 6,7,8 or 9 from 5 ' end;

Sense strand comprises 4,5 or 62 ' 5 ' continuous Nucleotide in 3 ' penultimate or 3 ' terminal position; And

In some embodiments of structure (A1) and/or structure (A2), (N) y comprises at least one the unconventional part that is selected from mirror nuclei thuja acid, 2 ' 5 ' ribonucleotides and TNA.In some embodiments, this unconventional part is mirror nuclei thuja acid.In multiple embodiments, this mirror nuclei thuja acid is selected from L-ribonucleotide (L-RNA) and L-deoxyribonucleotide (L-DNA).In preferred embodiments, this mirror nuclei thuja acid is L-DNA.In certain embodiments, sense strand the 9th or 10 (from 5 ' ends) comprise unconventional part.In preferred embodiments, sense strand comprises unconventional part at the 9th (from 5 ' end).In some embodiments, sense strand is that 19 Nucleotide are long, and comprises 4,5 or 6 continuous unconventional parts at the 15th (from 5 ' end).In some embodiments, sense strand comprises 42 ' 5 ' continuous ribonucleotides at the 15th, 16,17 and 18.In some embodiments, sense strand comprises 52 ' 5 ' continuous ribonucleotides at the 15th, 16,17,18 and 19.In multiple embodiments, sense strand further comprises Z '.In some embodiments, Z ' comprises C3OH part or C3Pi part.

In structure (A1) and/or some embodiments (A2), (N) y comprises at least one unconventional part, and described unconventional part is selected from mirror nuclei thuja acid or joins the Nucleotide of adjacent nucleotide by phosphate bond between 2 '-5 ' Nucleotide to.In some embodiments, this unconventional part is mirror nuclei thuja acid.In multiple embodiments, this mirror nuclei thuja acid is selected from L-ribonucleotide (L-RNA) and L-deoxyribonucleotide (L-DNA).In preferred embodiments, this mirror nuclei thuja acid is L-DNA.

In some embodiments of structure A1, (N ') y comprises at least one L-DNA part.In some embodiments, x=y=19 and (N ') y are made up of 1-17 position and the unmodified ribonucleotide of the 19th and a L-DNA of 3 ' penultimate (the 18th).In other embodiments, x=y=19 and (N ') y are made up of two continuous L-DNA Nucleotide of 1-16 position and the 19th 's unmodified ribonucleotide and 3 ' penultimate (the 17th and 18).In multiple embodiments, unconventional part is for being incorporated into the Nucleotide of adjacent nucleotide by phosphate bond splice grafting between 2 '-5 ' Nucleotide.According to multiple embodiments, (N ') y comprises 2,3,4,5 or 6 continuous ribonucleotides that connect by 2 '-5 ' internucleotide linkage at 3' end.In one embodiment, at four continuous kernel sugar nucleotides of the 3 ' end of (N ') y by three 2 '-5 ' phosphodiester keyed engagement.In one embodiment, at five continuous kernel sugar nucleotides of the 3 ' end of (N ') y by four 2 '-5 ' phosphodiester keyed engagement.In some embodiments, the wherein one or more formation 2 '-5 ' phosphodiester bonds in 2 '-5 ' ribonucleotide, it is sugar-modified that Nucleotide also comprises 3 '-O-methyl (3 ' OMe).In some embodiments, to comprise 3 ' OMe sugar-modified for the 3 ' terminal nucleotide of (N ') y.In certain embodiments, x=y=19 and (N ') y comprise two or more continuous nucleotides at the 15th, 16,17,18 and 19, and these Nucleotide are by being keyed onto adjacent nucleotide between 2 '-5 ' Nucleotide.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide comprises ribonucleotide.In preferred embodiments, between 2 '-5 ' Nucleotide, key is key between phosphodiester Nucleotide.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide comprises 3 ' deoxyribonucleotide or 3 ' methoxyl group Nucleotide.In multiple embodiments, the ribonucleotide that forms key between 2 '-5 ' Nucleotide comprises 3 ' deoxyribonucleotide or 3 ' methoxyl group ribonucleotide.In some embodiments, x=y=19 and (N ') y comprise the Nucleotide by being keyed onto adjacent nucleotide between 2 '-5 ' Nucleotide between 15-16,16-17 and 17-18 position or between 16-17,17-18 and 18-19 position.In some embodiments, x=y=19 and (N ') y comprise the Nucleotide by being keyed onto adjacent nucleotide between 2 '-5 ' Nucleotide between 16-17 and 17-18 position or between 17-18 and 18-19 position or between 15-16 and 17-18 position.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide comprises ribonucleotide.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide is ribonucleotide.In other embodiments, the pyrimidine ribonucleotide (rU, rC) in (N ') y is passed the ribonucleotide that is keyed onto adjacent ribonucleotide between 2 '-5 ' Nucleotide and replaces.

In some embodiments of structure (A2), (N) y comprises at least one L-DNA part.In some embodiments, x=y=18 and N2-(N ') y is made up of 1-17 position and the unmodified ribonucleotide of the 19th and a L-DNA of 3 ' penultimate (the 18th).In other embodiments, x=y=18 and N2-(N ') y is made up of two continuous L-DNA of 1-16 position and the 19th 's unmodified ribonucleotide and 3 ' penultimate (the 17th and 18).In multiple embodiments, unconventional part is for joining the Nucleotide of adjacent nucleotide to by phosphoric acid ester bonding between 2 '-5 ' Nucleotide.According to multiple embodiments, N2-(N ') y comprises 2,3,4,5 or 6 continuous ribonucleotides that connect by 2 '-5 ' internucleotide linkage at 3' end.In one embodiment, four continuous ribonucleotides of the 3 ' end of N2-(N ') y are by three 2 '-5 ' phosphodiester keyed engagement, and wherein forming one or more in 2 ' of 2 '-5 ' phosphodiester bond-5 ' ribonucleotide, also to comprise 3 '-O-methyl (3 ' OMe) sugar-modified.In some embodiments, to comprise 2 ' OMe sugar-modified for 3 ' the end ribonucleotide of N2-(N ') y.In certain embodiments, x=y=18 and N2-(N ') y comprises two or more continuous nucleotides at the 15th, 16,17,18 and 19, and these Nucleotide are by being keyed onto adjacent nucleotide between 2 '-5 ' Nucleotide.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide comprises 3 ' deoxyribonucleotide or 3 ' methoxyl group Nucleotide.In multiple embodiments, the ribonucleotide that forms key between 2 '-5 ' Nucleotide comprises 3 ' deoxyribonucleotide or 3 ' methoxyl group ribonucleotide.In some embodiments, x=y=18 and N2-(N ') y comprises the Nucleotide by being keyed onto adjacent nucleotide between 2 '-5 ' Nucleotide between 16-17 and 17-18 position or between 17-18 and 18-19 position or between 15-16 and 17-18 position.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide comprises ribonucleotide.In multiple embodiments, the Nucleotide that forms key between 2 '-5 ' Nucleotide is ribonucleotide.In other embodiments, the pyrimidine ribonucleotide (rU, rC) in (N ') y comprises the ribonucleotide by being keyed onto adjacent ribonucleotide between 2 '-5 ' Nucleotide.

In structure (A1) and/or other embodiments (A2), (N ') y comprises 1-8 the ribonucleotide of modifying, and the ribonucleotide of wherein modifying is ribodesose (DNA) Nucleotide.In certain embodiments, (N ') y comprises 1,2,3,4,5,6,7 or maximum 8 DNA parts.

In the preferred embodiment of the invention, inhibitor provided herein is double chain oligonucleotide that synthesize, chemically modified (for example dsRNA) compound, and it is selected from: lower HES1 and express and comprise the right double chain oligonucleotide of oligonucleotide that is selected from Table I; Lowering HES5 expresses and comprises the right double chain oligonucleotide of oligonucleotide that is selected from Table II; Lowering HEY2 expresses and comprises the right double chain oligonucleotide of oligonucleotide that is selected from Table III; Lowering CDKN1B expresses and comprises the right double chain oligonucleotide of oligonucleotide that is selected from Table IV; Lowering NOTCH1 expresses and comprises the right double chain oligonucleotide of oligonucleotide that is selected from Table V.Table I-V provides hereinafter.

The selected HES1dsRNA of Table I

The selected HES5dsRNA of Table II

The selected HEY2dsRNA of Table III

The selected CDKN1B of Table IV (p27) duplex

The selected NOTCH1dsRNA of Table V

In some embodiments of combination, composition and method, double chain oligonucleotide molecule comprises and is selected from the right sense strand of the oligonucleotide shown in Table I-V and antisense strand.Except as otherwise noted, equal from 5 ' to 3 ' (5 '-3 ') counting otherwise along all positions of sense strand or antisense strand.

In some embodiments, double chain oligonucleotide comprises the specific sense strand shown in SEQ ID NO:23-1495 or 26667-26706 (HES1), SEQ ID NO:1496-2703 or 26707-26732 (HES5), SEQ ID NO:13004-16621 or 26779-26788 (HEY2), SEQ ID NO:7444-10533 or 26867-26900 (CDKN1B) or SEQ ID NO:16622-26666 or 26901-26912 (NOTCH1) and specific antisense strand.

In some embodiments, double chain acid molecule has following structure:

Wherein each " │ " represents the base pairing between ribonucleotide;

Wherein each X is any one in A, C, G, U and is ribonucleotide unmodified or that modify, deoxyribonucleotide or unconventional part unmodified or that modify independently;

Wherein each of Z and Z ' has an independent existence or does not exist, if but while existence, be 1-5 continuous nucleotide or non-nucleotide part or its combination of the covalently bound 3 ' end at its existing chain independently; And

Wherein z " can exist or not exist, if but while existence, be the end-blocking part of the covalently bound 5 ' end at sense strand.

In preferred embodiments, the ribonucleotide that double chain oligonucleotide molecule comprises modification and unconventional part.

chemically modified

All analogues of Nucleotide/oligonucleotide or modification all can be used for embodiment of the present invention, and prerequisite is described analogue or modifies and can produce significant impact to the function of Nucleotide/oligonucleotide.Nucleotide can be selected from naturally occurring or synthetic modified base.Naturally occurring base comprises VITAMIN B4, guanine, cytosine(Cyt), thymine and uracil.The modified base of Nucleotide is described herein.

In addition, prepare the analogue of polynucleotide, the structure of wherein one or more Nucleotide fundamentally changes and is more suitable for as treatment or experiment reagent.The example of nucleotide analog is peptide nucleic acid(PNA) (PNA), and wherein ribodesose (or ribose) phosphate backbone in DNA (or RNA) is substituted to the similar polyamide skeleton being present in peptide.Show, the resistance to enzyme liberating of PNA analogue, and have in longer body and vitro stability.Other modifications that can carry out oligonucleotide (for example comprise main polymer chain, ring-type main chain, acyclic main chain, thiophosphoric acid-D-ribose main chain, three ester main chains, thioic acid sulfoacid main chain, 2 '-5 ' bridge joint main chain, artificial nucleic acid, morpholino nucleic acid, lock nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), Arabinoside and mirror nuclei thuja acid, beta-l-2 '-deoxy nucleosides, instead of β-D-deoxynucleoside).The example of the dsRNA molecule that comprises LNA Nucleotide is at the people such as Elmen (NAR 2005,33 (1): have disclosed 439-447).

The nucleic acid compound that can be used for method disclosed herein, composition and combination can use one or more inverted Nucleotide synthetic, such as inverted thymidine or the VITAMIN B4 that causes are (referring to people such as such as Takei, 2002, JBC 277 (26): 23800-06).

Term " unconventional part " refers to deoxyribonucleotide, mirror nuclei thuja acid, the non-base pairing nucleotide analog of dealkalize base ribose part, dealkalize base ribodesose part, deoxyribonucleotide, modification and joins the Nucleotide of adjacent nucleotide by phosphate bond between 2 '-5 ' Nucleotide to as used herein; C3, C4, C5 and C6 part; The nucleic acid of bridge joint, comprises the nucleic acid of LNA and ethylidene bridge joint.

Term " end-blocking part " comprises dealkalize base ribose part as used herein, and the modification of dealkalize base ribose and dealkalize base ribodesose part, comprises 2 ' O alkyl modified; Inverted dealkalize base ribose and dealkalize base ribodesose part and modification thereof; C6-imino--Pi; Comprise the mirror nuclei thuja acid of L-DNA and L-RNA; 5 ' OMe Nucleotide; With comprise 4', the nucleotide analog of 5'-methylene radical Nucleotide; 1-(the red furyl glycosyl of β-D-) Nucleotide; 4'-thio nucleotides, homocyclic nucleus thuja acid; 5'-aminoalkyl group phosphoric acid ester; 1,3-diamino-2-propyl phosphate, 3-Aminopropyphosphinic acid ester; The amino hexyl phosphoric acid ester of 6-; The amino 1-isobutyl-3,5-dimethylhexylphosphoric acid of 12-; Hydroxypropyl phosphoric acid ester; 1,5-anhydrohexitol Nucleotide; α-Nucleotide; Soviet Union's-penta furyl glycosyl Nucleotide; Acyclic 3', the 4'-Nucleotide that breaks; 3,4-dihydroxyl butyl Nucleotide; 3,5-dihydroxyl amyl group Nucleotide, the inverted dealkalize base section of 5'-5'-; BDO phosphoric acid ester; 5'-amino; With bridge or without bridge methylphosphonate and 5'-sulfydryl part.

Dealkalize base ribodesose part comprises for example dealkalize base ribodesose-3 '-phosphoric acid ester, 1,2-dideoxy-D-RIBOSE-3-phosphoric acid ester, Isosorbide-5-Nitrae-anhydrous-DRL-3-phosphoric acid ester.Inverted dealkalize base ribodesose part comprises inverted ribodesose dealkalize base, 3 ', 5 ' inverted ribodesose dealkalize base 5 '-phosphoric acid ester.

" mirror image " Nucleotide is the Nucleotide contrary with the chirality of naturally occurring or conventional Nucleotide, that is, and and the mirror image of naturally occurring (D-Nucleotide) (L-Nucleotide).Nucleotide can be ribonucleotide or deoxyribonucleotide, and can comprise at least one sugar, base and/or backbone modifications.U.S. Patent number 6,602,858 disclose the nucleic acid catalyst that comprises at least one L-Nucleotide replacement.Mirror nuclei thuja acid comprises for example L-DNA (L-ribodesose adenosine-3 '-phosphoric acid ester (mirror image dA), L-ribodesose cytidine-3 '-phosphoric acid ester (mirror image dC), L-ribodesose guanosine-3 '-phosphoric acid ester (mirror image dG), L-ribodesose thymidine-3 '-phosphoric acid ester (mirror image dT)) and L-RNA (L-ribose adenosine-3 '-phosphoric acid ester (mirror image rA), L-ribose cytidine-3 '-phosphoric acid ester (mirror image rC), L-ribose guanosine-3 '-phosphoric acid ester (mirror image rG), L-5-ribosyl uracil-3 '-phosphoric acid ester (mirror image dU)).

In the multiple embodiments of structure A1 or structure A2, Z and Z ' do not exist.In other embodiments, Z or Z ' exist.In some embodiments, each of Z and/or Z' comprises C2, C3, C4, C5 or C6 moieties independently, be optionally C3[propane,-(CH2) 3-] part or derivatives thereof, comprise the phosphodiester derivative (" C3Pi ") of propyl alcohol (C3-OH/C3OH), propylene glycol and propylene glycol.In preferred embodiments, each of Z and/or Z' comprises two hydrocarbon parts, and is C3Pi-C3OH or C3Pi-C3Pi in some instances.The key covalency that each C3 is preferably based on phosphorus by covalent linkage is conjugated to adjacent C3.In some embodiments, the key based on phosphorus is thiophosphatephosphorothioate, phosphonoacetic acid ester or phosphodiester bond.

In specific embodiments, at least one double chain oligonucleotide has structure A1, and x=y=19 and Z comprise at least one C3 alkyl and overhang.In specific embodiments, at least one double chain oligonucleotide has structure A2, and x=y=18 and Z comprise at least one C3 alkyl and overhang.In some embodiments, C3-C3 overhangs and amounts to valency and be attached to the 3 ' end of (N) x or (N ') y via covalent bonding preferably phosphoric acid diester linkage.In some embodiments, the bonding between a C3 and the 2nd C3 is phosphodiester bond.In some embodiments, 3 ' non-nucleotide is overhang for C3Pi-C3Pi.In some embodiments, 3 ' non-nucleotide is overhang for C3Pi-C3Ps.In some embodiments, 3 ' non-nucleotide is overhang for C3Pi-C3OH (OH is hydroxyl).In some embodiments, 3 ' non-nucleotide is overhang for C3Pi-C3OH.

In multiple embodiments, moieties comprises alkyl derivative, comprises C3 alkyl, C4 alkyl, C5 alkyl or the C6 moieties of Chinese terminal hydroxyl, terminal amino group or terminal phosphate group.In some embodiments, moieties is C3 alkyl or C3 alkyl derivative part.In some embodiments, C3 moieties comprises propyl alcohol, propyl phosphate, propyl dithiocarbamate phosphoric acid ester or its combination.C3 moieties can be covalently bound to the 3 ' end of (N ') y and/or (N) 3 ' end of x via phosphodiester bond.In some embodiments, moieties comprises propyl alcohol, propyl phosphate, propyl dithiocarbamate phosphoric acid ester.In some embodiments, each of Z and Z ' is multiple independently selected from propyl alcohol, propyl phosphate, propyl dithiocarbamate phosphoric acid ester, its combination or its, especially 2 or 3 covalently bound propyl alcohol, propyl phosphate, propyl dithiocarbamate phosphoric acid ester or its combinations.In some embodiments, each of Z and Z ' is independently selected from propyl phosphate, propyl dithiocarbamate phosphoric acid ester, propyl group phosphorus-propyl alcohol; Propyl group phosphorus-propyl dithiocarbamate phosphoric acid ester; Propyl group phosphorus-propyl phosphate; (propyl phosphate) 3, (propyl phosphate) 2-propyl alcohol, (propyl phosphate) 2-propyl dithiocarbamate phosphoric acid ester.Any propane or propyl alcohol are puted together part and all can be included in Z or Z '.

Exemplary 3 ' end non-nucleotide part is as follows:

indication

Molecule disclosed herein and composition can be used for treating ear disease and obstacle, and other diseases as herein described and illness.

people's ear

People's ear is made up of three main structure integral parts: external ear, middle ear and inner ear, and they play a role sound wave to be changed into the Nerve impulse that is delivered to brain together, and in brain, they are as sound and perceived arriving.Inner ear also contributes to maintain balance.

The dissection of middle ear and inner ear is that those of ordinary skill in the art know (referring to the Atlas of Sensory Organs:Functional and Clinical Analysis being for example incorporated herein by reference, Andrs Csillag, Humana Press (2005), 1-82 page).In brief, inner ear is made up of the chamber of ear-drum and a less air filling, and this chamber comprises that a succession of totally three ossiculum heads that are called otosteon form, and ear-drum is connected to inner ear by this otosteon.

The complex construction that inner ear (getting lost) is made up of cochlea, cochlea is the organ of hearing and vestibular system, the organ of balance.Vestibular system is by determining the saccule and utricle of topognosia and contributing to the semicircular duct that maintains balance to form.

Cochlea holds corti's organ, and it is partly made up of the sensory cell (being called " inner ear hair cells " or " hair cell ") of about 20,000 specializations.These cells have the tiny projection (cilium) that extends into cochlea liquid.The sound vibration that otosteon from middle ear is delivered to the oval window in inner ear causes cochlea liquid and cilium vibration.Hair cell in cochlea different piece vibrates in response to different sound frequencies, and vibration is changed into and sends to the Nerve impulse of brain to process and to understand.Inner ear hair cells (IHC) by inner ear supporting cell around.Sustenticular cell is arranged under inner ear Sensory hair cell, at least in part around and physical support described in hair cell.The representative example of sustenticular cell comprises interior retinal rod (pillar cell), outer retinal rod (pillar cell), inner phalangeal cell, external phalangeal cell (Deiters'cells, Held cell, Hensen cell, Claudius cell, Boettcher cell, interdental cell and listen tooth (Huschke listens tooth).

Spiral ganglion is a group neurocyte that the performance of sound is sent to brain from cochlea.The cell paste of Spiral ganglion neuron is present in cochlea spirane structure and is a part for central nervous system.Their dendron forms cynapse with the substrate of hair cell and contacts, and their aixs cylinder bundles the sense of hearing part that forms the 8th cranial nerve (vestibulocochlear nerve).

hearing loss

Auditory hair cell relates to the susceptor that is arranged in cochlea corti's organ of sensing sound.Cochlear hair cell is divided into two kinds of types different in anatomy and function: external ear hair cell and inner ear hair cells.Acoustic information is changed into electrical signal by auditory hair cell, and these electrical signal send to brain and process via nerve fiber.

Be arranged in inner ear (Utriculus, sacculus, ampulla) vestibule vestibular hair cells sensing head position variation and this information is communicated to brain to contribute to maintain posture and the eye position of balance.

In the situation that not there is not auditory hair cell, sound wave can not change into nerve signal, and hearing defect then occurs, and for example, acouesthesia reduces, that is, and and sensorineural hearing loss.In the situation that not there is not vestibular hair cells, balance is damaged then to be occurred.

Although acoustic reflex exists provide protection, large noise can damage and destroy hair cell.Irreversible Hair Cell Death is caused by the metabolism or the biochemical change that relate in the hair cell of reactive oxygen species (ROS).Be exposed to some drugs and continue to be exposed to large noise and especially can cause progressive injury, finally causing tinnitus and hearing loss.

Acquired hearing loss can cause because of many factors, comprise be exposed to harmful noise level, be exposed to ototoxic drug (such as cis-platinum and aminoglycoside antibiotics) and aging.

The U.S. Patent number 11/655,610 of authorizing assignee of the present invention relate to by suppress generally to urge apoptogene especially p53 treat the method for dysaudia.International Patent Publication No. W WO2005/119251 relates to the deaf method for the treatment of.International Patent Publication No. W WO/2005/055921 relates to the foam composition that is used for the treatment of ear's obstacle.U.S. Patent number 7,087,581 relate to the method for the treatment of disease of inner ear and obstacle.Transfer present assignee and be incorporated to PCT publication No. WO 2009/147684 herein with way of reference entirety and disclose some compound and the composition that are used for the treatment of ear's obstacle and disease.

ear's obstacle

The disclosure relates in particular to and can be used for treating composition, combination and the method for suffering from various ears obstacle or the patient in various ears obstacle risk.Ear's obstacle comprises the hearing loss for example being caused by ear toxin, excessive noise or aging.Middle ear and inner ear obstacle produce many identical symptoms, and the obstacle of middle ear can affect inner ear, and vice versa.

Gone out outside hearing loss, ear's obstacle also comprises myringitis, and a kind of eardrum being caused by multiple virus and bacterium infects; The temporal bone fracture for example causing due to head impact; Auditory nerve knurl (acoustic tumor, acoustic nerve neurilemmoma, vestibular nerve schwannomas, eighth nerve knurl).

In multiple embodiments, method disclosed herein, combination and composition can be used for treating the various illnesss of hearing loss.Be not bound by theory, hearing loss can be because of (people such as Zhang, Neuroscience 2003.120:191-205 due to apoptosis inner ear hair cells infringement or loss; The people such as Wang, J.Neuroscience 23 ((24): 8596-8607), wherein this infringement or loss are caused by the ototoxicity of infection, physical abuse, loud (noise), aging (presbyacusis) or chemical induction.

Under background disclosed herein, so-called " ear toxin " refers to by the activity of the neural system phonoreceptor integral part that its chemical action is damaged, damaged or inhibition is relevant to hearing, the then material of hearing damage (and/or balance).In the context of the present invention, ototoxicity comprises the deleterious effect of Ear hair cell.Cause the ototoxicity agent of hearing loss to include but not limited to tumour agent, for example, such as vincristine(VCR), vinealeucoblastine(VLB), cis-platinum and cis-platinum compounds, taxol and taxol compounds, dideoxy compound, didanosine; Alcohol; Metal; The industrial pollutants that relate in occupation or environmental exposure; Food or drug contamination thing; For example, with VITAMIN or the curative drug of overdose, microbiotic, such as penicillin or paraxin, and heavy dose of vitamin A, D or B6, salicylate, quinine and loop diuretic.So-called " being exposed to ototoxicity agent " refers to and ototoxicity agent is exposed to or contacts Mammals.Being exposed to ototoxicity agent can be by directly using and occurs, for example for example, occur by absorbing or use food, medicine or therapeutical agent (chemotherapeutics), occur by accidental pollution, or for example, occur by environmental exposure (air or water expose).Conventionally, treat to prevent or alleviate ototoxicity, especially because of or estimate the ototoxicity causing because of administering therapeutic medicine.Preferably, after exposure, comprise immediately the composition of the chemically modified siRNA compound of the present invention for the treatment of significant quantity, to prevent or to alleviate ototoxicity effect.More preferably, by before using ototoxic drug or being exposed to ear toxin or use concomitantly pharmaceutical composition of the present invention and treatment is prophylactically provided.Be incorporated herein by reference The Merck Manual of Diagnosis and Therapy, the 14th edition, (1982), Merck Sharp & Dome Research Laboratories, N.J. the 196th, 197,198 and 199 chapters, and corresponding chapters and sections in nearest the 16th edition, comprise the 207th and 210 chapters of the description and the diagnosis that relate to hearing and disequilibrium.

Therefore, in one aspect, the invention provides and be used for the treatment of the preferred mankind of Mammals to prevent, to alleviate or to treat hearing loss, obstacle or unbalance, method, combination and the pharmaceutical composition of the hearing illness that preferably ear toxin causes, mode is to have the administration needing to comprise combination or the composition of target gene inhibitor as disclosed herein to this treatment.Some embodiments relate to the method that is used for the treatment of dysaudia or damage, and wherein ototoxicity causes because of the ototoxic drug of administering therapeutic significant quantity.Typical ototoxic drug is chemotherapeutics, for example antineoplastic agent, and microbiotic.Other possible material standed fors comprise loop diuretic, quinine or quinine compounds, PDE-5 inhibitor and salicylate or salicylate compounds.

Ototoxicity is the dose limitation side effect of antibiotic administration.Accept 1 gram of patient who continues to exceed 1 week 4% to 15% every day the hearing loss that can survey occurs, if continual cure can slowly become more serious and can cause permanent deafness completely.Ototoxicity aminoglycoside antibiotics includes but not limited to Liu Suanyan NEOMYCIN SULPHATE, paromycin, ribostamycin, Lividomycin, kantlex, amikacin, tobramycin, Viothenate, gentamicin, sisomicin, netilmicin, Streptomycin sulphate, dibekacin, fortimicin and Vibriomycin or its combination.Specific microbiotic comprises Soframycin, kanamycin A, kanendomycin, gentamicinC1, Gentamicin C1a and gentamicinC2 and knownly has an especially analogue of ototoxicity and renal toxicity of serious toxicity, these toxicity can reduce the availability of this type of biocide (referring to Goodman and Gilman's The Pharmacological Basis of Therapeutics, the 6th edition, the people such as A.Goodman Gilman edit; Macmillan Publishing Co., Inc., New York, 1169-71 page (1980)).

Ototoxicity is also the serious dose limitation side effect of carcinostatic agent.The agent of ototoxicity tumour includes but not limited to vincristine(VCR), vinealeucoblastine(VLB), cis-platinum and cis-platinum compounds and taxol and taxol compounds.Cis-platinum compounds especially comprise carboplatin ( ), four platinum, oxaliplatin, oxaliplatin liposome (aroplatin) and cis-platinum be the chemotherapeutics based on platinum.

The diuretic(s) with known ototoxicity side effect especially " loop " diuretic(s) includes but not limited to furosemide, ethylacrylic acid and mercurial.

Ototoxicity quinine includes but not limited to be generally used for the synthetic substitute of the quinine for the treatment of malaria.In some embodiments, dysaudia is the side effect of 5 type phosphodiesterase (PDE-5) inhibitor, described inhibitor comprise Virga ( ), Vardenafil ( ) and Tadalafei (Cialis).

Such as the salicylate of acetylsalicylic acid because of its anti-inflammatory, pain relieving, antipyretic and anti thrombotic action and be the most frequently used curative drug.Unfortunately, they also have ototoxicity side effect.They cause tinnitus (tinnitus) (" tinnitus (ringing in the ears) ") and temporary hearing loss conventionally.In addition,, if use this medicine with high dosage for a long time, hearing loss meeting becomes lasting and irreversible.

In some embodiments of method provided in this article, experimenter suffers from the Mammals that infects and treat by using aminoglycoside antibiotics.Method disclosed herein is by alleviating or preventing that the hearing loss that the ear toxin relevant to microbiotic causes from improving lapsing to of this type for the treatment of.

Method as herein described, combination and pharmaceutical composition also can effectively be treated sense of hearing wound or mechanical trauma, preferably cause the sense of hearing or the mechanical trauma of inner ear hair cells loss.For more serious exposure, damage can develop into from the loss of adjacent sustenticular cell the destruction completely of corti's organ.The death of sensory cell can cause the loss of gradual Wallerian degeneration and elementary acoustic fibers.Method provided in this article, combination and composition can be used for the sense of hearing wound that treatment causes the every day higher than 85 decibels in great sound or long-term exposure loudly because of single exposure; Be used for the treatment of mechanicalness inner ear wound, for example, because mechanism being inserted due to inner ear; Or prevent or at utmost reduce the infringement of the Ear hair cell relevant to operation.

The hearing loss of another kind of type is presbyacusis, and it is the hearing loss occurring gradually when older in most of individualities.The about 30-35% age is experienced hearing loss between the grownup between 65-75 year and 40-50% age 75 years old and above people.Method disclosed herein, combination and composition can be used for inner ear obstacle that prevention, reduction or treatment are relevant to presbyacusis and incidence and/or the seriousness of hearing loss.

sense of hearing wound

Sense of hearing wound is the hearing loss type that long-term exposure causes in high noisy.Do not wish to be bound by theory, be exposed to high noisy and cause the hair cell Reduced susceptibility on cochlea.For more serious exposure, damage can develop into from the loss of adjacent sustenticular cell the destruction completely of corti's organ.The death of sensory cell can cause the loss of gradual Wallerian degeneration and elementary acoustic fibers.The combination, pharmaceutical composition and the method that can be used for alleviating the hearing loss causing because of sense of hearing wound are especially disclosed herein.In some embodiment of method, combination and composition, the dsRNA molecule of target HES1, HES5 and HEY2 is used for the treatment of or prevents to the experimenter's who is exposed to sense of hearing wound sense of hearing wound.In some embodiment of method, combination and composition, the dsRNA molecule of target CDKN1B, NOTCH1 and HEY2 is used for the treatment of or prevents to the experimenter's who is exposed to sense of hearing wound sense of hearing wound.

In certain embodiments, provide treatment to suffer from ear's obstacle or the experimenter's in ear's obstacle risk method herein, it comprises the duct topical application pharmaceutical composition to experimenter ear, the inhibitor of the gene that the target of its amount that comprises effective treatment experimenter is relevant to described obstacle is such as dsRNA inhibitor, with pharmaceutically acceptable vehicle or its mixture, thereby treat described experimenter.In certain embodiments, provide treatment to suffer from ear's obstacle or the experimenter's in ear's obstacle risk method herein, it comprises that duct to experimenter ear is through tympanum drug administration composition, the inhibitor of the gene that the target of its amount that comprises effective treatment experimenter is relevant to described obstacle is such as oligonucleotide inhibitor, with pharmaceutically acceptable vehicle or its mixture, thereby treat described experimenter.In one embodiment, pharmaceutical composition being cut to dilatation via posterior semicircular duct sends.In one embodiment, pharmaceutical composition is sent as ear drop.In another embodiment, pharmaceutical composition is sent by pump.

In some embodiments, be inclined to a side and the ear for the treatment of upwards time at experimenter's head, pharmaceutical composition is applied to duct.In some embodiments, use ear drop container to be applied to ear pharmaceutical composition, for example, use the dropper of every of 10-100 microlitre for example, or fuse.

In some embodiments, ear's obstacle relates to the hearing loss that chemical causes; The hearing loss for example especially causing because of cis-platinum and analogue, aminoglycoside antibiotics, quinine and analogue thereof, salicylate and analogue, PDE5 type (PDE5) inhibitor or loop diuretic.In some embodiments, ear's obstacle refers to the hearing loss that noise causes.In other embodiments, ear's obstacle is age related hearing loss.

Be not bound by theory, the inhibition of HES1, HES5, HEY2, CDKN1B or NOTCH1 causes regeneration or the protection of inner ear tragus (sensation) cell, the increase of optionally expressing via Atoh1.Method, composition and combination can be used for treatment, improve or prevention wherein needs to promote inner ear supporting cell in cochlea or any disease, obstacle or the damage of external ear hair cell or inner ear hair cells propagation as disclosed herein.In multiple embodiments, method provided in this article, composition and combination can be used for treating hearing and disequilibrium, the hearing loss that the hearing loss causing such as, but not limited to ear toxin, the hearing loss relevant to Meniere and wound cause, such as sense of hearing wound and pressure wound, be included in impact and surgical operation in inner ear and/or middle ear.

vestibular system disease and obstacle

In multiple embodiments, nucleic acid compound disclosed herein and pharmaceutical composition can be used for treatment affects obstacle and the disease of vestibular system, in these obstacles and disease, the expression of HES1, HES5 and HEY2 or CDKN1B, NOTCH1 and HEY2 is harmful to, for example Meniere.Vestibular sense system in the most of Mammalss that comprise the mankind contributes to balance, and contributes to sense space orientation and stability.Together with cochlea, it forms the lost of inner ear.Vestibular system comprises two integral parts: the semicircular duct system that instruction rotatablely moves, and the linear otoconia accelerating of instruction.

The first attack relevant to Meniere is dizzy and carrying out property hearing loss is made us weak person's character.Current therapy is not yet obtaining successfully aspect prevention neuronal degeneration and relevant hearing loss.In ingestion in patient with Meniere ', will avoid inner ear neurone (comprising vestibulocochlear nerve) infringement and/or the regeneration of induction vestibulocochlear nerve and thereby alleviate or prevent that the therapeutic treatment of hearing loss from will be very desirable.

In some aspects with embodiment in, combination provided in this article, composition, method, commercial package and test kit can be used for treatment in Meniere risk or suffer from the experimenter of Meniere.

In a word, still there is not the effective treatment pattern for preventing and/or treating illness disclosed herein.Especially there is selectivity target for want of and the shortcoming of the serious side effects that causes in available treatment, and therefore still needs novel composition and the methods for the treatment of of exploitation for these objects.

In multiple embodiments, combination provided in this article and pharmaceutical composition can be used for treatment or prevent multiple disease, obstacle and the damage that affects ear, such as, but not limited to below disclosed disease, obstacle and damage.Be not bound by theory, it is believed that combination/composition disclosed herein prevents the interior polytype necrocytosis of ear and/or promotes the sustenticular cell in inner ear to be divided into ear sensory cell.

pharmaceutical composition

The invention provides expression for lowering HES1, HES5 and HEY2 or for lowering composition, combination and the method for expression of CDKN1B, HEY2 and NOTCH1.In certain embodiments, said composition, combination and method are used small nucleic acids molecule, such as short interfering nucleic acid (siNA), RNA interfering (RNAi), short interfering rna (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecule, they can mediate downward or the mediation of HES1, HES5, HEY2, CDKN1B or NOTCH1 genetic expression and disturb for the RNA of HES1, HES5, HEY2, CDKN1B or NOTCH1 genetic expression.

Although molecule disclosed herein can be used as original chemical, preferably, be shown as pharmaceutical composition.Therefore, the invention provides one or more or its pharmacology salt comprising in dsRNA molecule disclosed herein, and the pharmaceutical composition of pharmaceutically acceptable carrier.Said composition can comprise the mixture of two or three different IPs acid compound.

Composition provided in this article, method and test kit can comprise independently or regulate in combination one or more nucleic acid molecule (for example dsRNA) and method: HES1, HES5, HEY2, CDKN1B or the NOTCH1 albumen of expression and/or the gene of encode HES1, HES5, HEY2, CDKN1B or NOTCH1 albumen of following albumen and gene, especially the maintaining and/or develop relevant albumen and/or gene of ear's dependency obstacle of, illness diseases related to HES1, HES5, HEY2, CDKN1B or NOTCH1 or obstacle.Provide the description to many aspects and embodiment with reference to exemplary gene HES1, HES5, HEY2, CDKN1B or NOTCH1.But, many aspects and embodiment also relate to other genes involveds, for example, such as homologue gene with transcribe variant and the polymorphism (single nucleotide polymorphism (SNP)) with some HES1, HES5, HEY2, CDKN1B or NOTCH1 gene-correlation.Therefore, many aspects also relate to embodiment the path mediating with HES1, HES5, HEY2, CDKN1B or the NOTCH1 of signal transduction or genetic expression and have other associated genes, and described path for example exists associated in disease as herein described, speciality or illness.Can use the target spot for these the other genes of methods analyst described in HES1, HES5, HEY2, CDKN1B or NOTCH1 gene herein.Therefore, can as described hereinly carry out, measure and measure the downward of other genes and this regulating effect of other genes.

The present invention also provides pharmaceutical composition, at least one of its amount that comprises effective downward HES1, HES5, HEY2, CDKN1B or NOTCH1 expression is covalently or non-covalently bonded to the compound of the present invention of one or more compounds of the present invention, and pharmaceutically acceptable carrier.The present invention also provides nucleic acid compound, and it is processed in cell by endogenous cell complex body, can be according to the oligoribonucleotide of method as herein described and embodiment use thereby produce one or more.

The present invention also provides pharmaceutical composition, its comprise pharmaceutically acceptable carrier and the compound that can use according to method disclosed herein in one or more, its amount effectively suppresses mankind HES1, HES5, HEY2, CDKN1B or the expression of NOTCH1 in cell, and described compound comprises substantially and the sequence of continuous sequence complementation that is selected from the sequence in HES1mRNA, HES5mRNA, HEY2mRNA, CDKN1B mRNA or NOTCH1mRNA.

Substantially the complementary complementarity that is greater than approximately 84% with another sequence that refers to.For example, in the duplex region being made up of 19 base pairs, mispairing produces 94.7% complementarity, and two mispairing produce approximately 89.5% complementarity, and 3 mispairing produce approximately 84.2% complementarity, thereby makes duplex region substantially complementary.Therefore, the substantially the same identity that is greater than approximately 84% with another sequence that refers to.

In addition, prevention is provided herein, treatment or delay experimenter dysaudia, the progress of hearing loss and/or disequilibrium, or prevent the method for ear (sensation) hair cell loss of inner ear, comprise compared with the control by HES1, HES5, HEY2, the expression inhibiting at least 20% of CDKN1B or NOTCH1 gene, at least 30%, at least 40%, preferably 50%, 60% or 70%, more preferably 75%, 80% or 90%, described method comprises corresponding gene mRNA transcript and HES1 inhibitor, HES5 inhibitor, HEY2 inhibitor, CDKN1B inhibitor or the contact of NOTCH1 inhibitor.

In multiple embodiments as herein provided, inhibitor is oligoribonucleotide compound.Composition disclosed herein, combination and method suppress/lower the expression of HES1, HES5, HEY2, CDKN1B or NOTCH1 gene, thereby suppress/lower to be selected from the inhibition/downward of the inhibition/downward of gene function, the inhibition of polypeptide/lower mediation mrna expression.

In certain embodiments, composition provided in this article, combination and method comprise (for example lowers HES1, HES5, HEY2, CDKN1B or NOTCH1 gene, by the mRNA encoding sequence of people HES1, HES5, HEY2, CDKN1B or the NOTCH1 of SEQ ID NO:1,2,10,7 or 11 examples) double-stranded short interfering nucleic acid (siNA) compound of expression, wherein nucleic acid molecule comprises approximately 18 to approximately 49 base pairs.

In some embodiments, nucleic acid disclosed herein can be used for suppressing the expression of HES1, HES5, HEY2, CDKN1B or NOTCH1 gene or HES1, HES5, HEY2, CDKN1B or NOTCH1 gene family, wherein said gene or gene family sequence consensus sequence homology.This type of homologous sequence can be identified as known in the art and in addition, for example, use sequence alignment.Can design with this type of homologous sequence of target nucleic acid molecule, for example, use perfect complementary sequence or for example, by mixing the non-standard base pair of the target sequence that can provide other, mispairing and/or wobble base pair.The in the situation that of qualification mispairing, can be for example, by non-standard base pair (, mispairing and/or wobble base) for generating the nucleic acid molecule of the more than a kind of gene order of target.In limiting examples, by the non-standard base pair such as UU and CC base pair for generate can target sequence nucleic acid molecule, thereby distinguish HES1, HES5, HEY2, CDKN1B or the NOTCH1 target of consensus sequence homology.Therefore, use an advantage of dsRNA disclosed herein to be to design single nucleic acid, to comprise and the nucleotide sequence of conservative nucleotide sequence complementation between homologous gene.In the method, can be by mononucleotide for suppressing the expression of more than a kind of gene, instead of carry out the different gene of target with more than a kind of nucleic acid.

Nucleic acid molecule can be used for to the conserved sequence of target corresponding to one or more gene families such as HES1, HES5, HEY2, CDKN1B or NOTCH1 family gene.Therefore, the nucleic acid molecule of the multiple HES1 of target, HES5, HEY2, CDKN1B or NOTCH1 target can provide the result for the treatment of of enhancing.In addition, can be by nucleic acid for characterize the path of gene function in multiple application.For example, can be by nucleic acid molecule for suppressing the activity of target gene at path, to determine the function of the gene not characterizing in gene function molecule, mRNA functional analysis or translation are analyzed.Can be by nucleic acid molecule for determining that the potential target gene path relating in various diseases and illness is with developing drugs.Can be by nucleic acid molecule for understanding the path in the genetic expression that for example ear's obstacle relates to.

At composition provided in this article, in the multiple embodiments of combination and method, nucleic acid compound suppresses HES1, HES5, HEY2, CDKN1B or NOTCH1 polypeptide, thereby suppress to be selected from the inhibition (it especially can be measured and study by enzymatic determination or with the combination of known action of natural gene/polypeptide) of function, (it especially can pass through Western trace for the downward of albumen or the inhibition of albumen, ELISA or immuno-precipitation are studied) and the inhibition of mrna expression (it especially can pass through Northern trace, quantitative RT-PCR, in situ hybridization or microarray hybridization are studied).

In certain embodiments, composition provided in this article, combination and method comprise the nucleic acid molecule having for the RNAi activity of HES1, HES5, HEY2, CDKN1B or NOTCH1RNA, and wherein nucleic acid molecule comprises and the sequence of any RNA complementation with HES1, HES5, HEY2, CDKN1B or NOTCH1 encoding sequence (such as in the sequence shown in SEQ ID NO:1,2,10,7 or 11).In another embodiment, nucleic acid molecule can have the RNAi activity for HES1, HES5, HEY2, CDKN1B or NOTCH1RNA, wherein this nucleic acid molecule comprises and the sequence of RNA complementation with variant HES1, HES5, HEY2, CDKN1B or NOTCH1 encoding sequence, for example not shown in SEQ ID NO:1,2,10,7 or 11 but be for example known in the art, to the generation of any obstacle disclosed herein (SNP) and/or maintain the sudden change in relevant HES1, HES5, HEY2, CDKN1B or NOTCH1 gene.Chemically modified is applicable to any nucleic acid construct disclosed herein as described herein.In another embodiment, nucleic acid molecule disclosed herein comprises and can interact also thereby the nucleotide sequence of downward or reticent HES1, HES5, HEY2, CDKN1B or NOTCH1 genetic expression with the nucleotide sequence of HES1, HES5, HEY2, CDKN1B or NOTCH1 gene, for example, wherein said nucleic acid molecule is by regulatory gene chromatin Structure or methylation patterns and prevent that the cell processes of genetic transcription from mediating the regulation and control of HES1, HES5, HEY2, CDKN1B or NOTCH1 genetic expression.

send and preparation

The inhibitor (for example, dsRNA molecule) that can use according to aspect disclosed herein and embodiment can be by being applied directly to pharmaceutical composition external ear, by through injection of tympanum, by pump or be delivered to experimenter's ear by ear drop.In some embodiments, pharmaceutical composition is applied to duct.Be delivered to ear and also can be described as ear and send, it comprises for example siRNA, penetration enhancers and pharmaceutically acceptable vehicle.

In multiple embodiments, can incite somebody to action inhibitor (for example nucleic acid molecule) as disclosed herein and be delivered to target tissue by directly apply the naked molecule of preparing together with carrier or thinner.

Term " naked nucleic acid " or " naked dsRNA " or " naked siRNA " refer to not containing the nucleic acid molecule that plays any delivery vehicle auxiliary, that promote or be conducive to the effect that enters cell (comprising virus sequence, virus particle, Liposomal formulation, liposome (lipofectin) or precipitation agent etc.).For example, the dsRNA in PBS is " naked dsRNA ".

In multiple embodiments, can by inhibitor disclosed herein (such as nucleic acid molecule) with play auxiliary, promote or be conducive to directly send or use together with the carrier of the effect that enters cell or thinner (comprising virus vector, virus particle, Liposomal formulation, liposome (lipofectin) or precipitation agent etc.).

Nucleic acid molecule can comprise delivery vehicle (comprising liposome), carrier and thinner and the salt thereof for being administered to experimenter, and/or can be present in pharmaceutically acceptable preparation.In some embodiments, dsRNA molecule disclosed herein is sent in Liposomal formulation and liposome (lipofectin) preparation etc., and can prepare by method well known to those skilled in the art.These class methods have disclosed in U.S. Patent number 5,593,972,5,589,466 and 5,580,859 for example, and these patents are incorporated herein by reference.

Develop specially for strengthening and improve delivery system that siRNA sends to mammalian cell (referring to the people such as such as Shen, FEBS Let.2003,539:111-114; The people such as Xia, Nat.Biotech.2002,20:1006-1010; The people such as Reich, Mol.Vision 2003,9:210-216; The people such as Sorensen, J.Mol.Biol.2003.327:761-766; The people such as Lewis, Nat.Gen.2002, the people such as 32:107-108 and Simeoni, NAR 2003,31,11:2717-2724).SiRNA recently successfully for the genetic expression that suppresses primate (referring to the people such as such as Tolentino, Retina 24 (4): 660).

Naked or preparation RNA molecule is sent especially by carrying out through injection of tympanum or the required compound that is mixed with ear drop by using to the optional inner ear of ear.The ear that comprises dsRNA with composition authorize in present assignee the U.S. Patent Publication that is incorporated herein by reference of entirety numbers 20110142917, have disclosed.

The polypeptide that is conducive to the nucleic acid to introduce required experimenter is known in this area, for example, for example, at the those polypeptides (melamine derivative described in U.S. Patent Application Publication No. 20070155658, such as 2,4,6-tri-guanidine radicals triazines and 2,4,6-triamido sarkosine trimeric cyanamide, poly arginine polypeptide, and comprise glutamine alternately and the polypeptide of asparagine residue).

Pharmaceutically acceptable carrier, solvent, thinner, vehicle, adjuvant and vehicle and implantation carrier generally refer to non-toxic solid or liquid filling agent, thinner or encapsulating material inertia, that do not react with activeconstituents of the present invention, and they comprise liposome and microballoon.The example that can be used for delivery system of the present invention sees U.S. Patent number 5,225, and 182,5,169,383,5,167,616,4,959,217,4,925,678,4,487,603,4,486,194,4,447,233,4,447,224,4,439,196 and 4,475,196.Many other these type of implants, delivery system and module are known to those skilled in the art.

In specific embodiments, use and comprise through tympanum and using.In another embodiment, use and comprise external application or topical application.The combination of creme, ointment for ear, solution, foam, mousse or above any one and delivery apparatus using compound as ear drop, for ear and using.The implant of compound is also useful.Prepare liquid form as drops or in order to apply continuously.Liquid composition comprises the aqueous solution, water or the oil suspension containing and do not contain organic cosolvent, the emulsion that contains edible oil and similar pharmaceutical vehicles.These compositions also can be injected through eardrum.Ear drop also can be called as ear drops.In preferred embodiments, ear drop is stayed in duct approximately 30 minutes to prevent that ear drop from spilling duct.It is therefore preferable that the experimenter who accepts ear drop by amesiality its head ear for the treatment of that makes upwards to prevent that ear drop from spilling duct.

Be used for the method for nucleic acid delivery molecule people such as Akhtar, Trends Cell Bio., 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, editor Akhtar, (1995), the people such as Maurer, Mol.Membr.Biol., 16:129-140 (1999); Hofland and Huang, Handb.Exp.Pharmacol., 137:165-192 (1999); With the people such as Lee, ACS Symp.Ser., 752:184-192 (2000); U.S. Patent number 6,395,713,6,235,310,5,225,182,5,169,383,5,167,616,4,959217,4.925,678; 4,487,603 and 4,486,194 and the people such as Sullivan, PCT WO94/02595, PCT WO 00/03683 and PCT WO 02/08754; And describe to some extent in U.S. Patent Application Publication No. 2003077829.These schemes can be used for sending any nucleic acid molecule substantially.Nucleic acid molecule can be administered to cell by several different methods well known by persons skilled in the art, these methods include but not limited to be encapsulated in liposome, by iontophoresis or by mixing other media thing such as Biodegradable polymer, hydrogel, cyclodextrin (referring to people such as such as Gonzalez, Bioconjugate Chem., 10:1068-1074 (1999); The people such as Wang, International PCT publication No. WO 03/47518 and WO 03/46185), Poly(D,L-lactide-co-glycolide (PLGA) and PLCA microballoon be (referring to for example U.S. Patent number 6,447,796 and U.S. Patent Application Publication No. 2002130430), in biodegradable nano capsule and bioadhesive microballoon, or by proteinaceous carrier (O ' Hare and Normand, International PCT publication No. WO 00/53722).Or, by direct injection or by using the combination of infusion pump local delivery nucleic acid/vehicle.Direct injection nucleic acid molecule of the present invention (be no matter vitreum interior, subcutaneous, through tympanum, intramuscular or intradermal) can use standard needle and syringe method carry out or by needleless technology people such as Conry, Clin.Cancer Res., the people such as 5:2330-2337 (1999) and Barry, those technology described in International PCT publication No. WO 99/31262 are carried out.Molecule of the present invention can be used as medicament.Medicament to a certain extent (preferably complete) prevents the symptom of morbid state, the generation that regulates described symptom or treatment or alleviates described symptom.In a specific embodiments of the present invention, can select external application and preparation capable of permeating skin.

Pharmaceutical composition disclosed herein and combination consider that according to good medical standard following aspect uses and administration: the clinical disease of individual patient, disease to be treated, site of administration and method, use other known factors of plan, patient age, sex, body weight and doctor.

In another embodiment, use and comprise external application or topical application, such as via ear drop or ointment.In limiting examples, the dsRNA compound of target HES1, HES5, HEY2, CDKN1B or NOTCH1 can be used for treatment and suffers from the experimenter that ear is damaged, and wherein dsRNA compound is sent to (for example ear drop or ointment) and is delivered to ear via outside.Nucleic acid molecule can be compound with cation lipid, be packaged in liposome or be otherwise delivered to target cell or tissue.Nucleic acid or nucleic acid complexes can be in the situation that mixing or do not mix biological polymer by direct cutaneous apply, through skin apply or inject and in in vitro or body mode locally apply to related tissue.The preferred oligonucleotide that can be used for generating dsRNA molecule discloses herein.

Delivery system can comprise the surface modification liposome that contains polyethyleneglycol lipid (PEG modification or long circulating liposomes or hidden liposome).These preparations provide increases the method that medicine is accumulated in target tissue.This class pharmaceutical carrier is by mononuclear phygocyte system (MPS or RES) opposing opsonification and elimination effect, thereby make blood circulation time can be longer and the tissue that increases entrapped drug expose the (people such as Lasic, Chem.Rev.1995,95,2601-2627; The people such as Ishiwata, Chem.Pharm.Bull.1995,43,1005-1011).

Nucleic acid molecule can be prepared or be compound together with following composition: polyaziridine (for example straight or branched PEI) and/or polyaziridine derivative, comprise for example polymine-polyoxyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polymine-polyoxyethylene glycol-tri--N-acetylgalactosamine (PEI-PEG-triGAL) derivative, the PEI of grafting, such as semi-lactosi PEI, cholesterol PEI, antibody derivatize PEI and polyoxyethylene glycol PEI (PEG-PEI) and derivative thereof (referring to people such as such as Ogris, 2001, AAPA Pharm Sci, 3,1-11; The people such as Furgeson, 2003, Bioconjugate Chem., 14,840-847; The people such as Kunath, 2002, Pharmaceutical Research, 19,810-817; The people such as Choi, 2001, Bull.Korean Chem.Soc., 22,46-52; The people such as Bettinger, 1999, Bioconjugate Chem., 10,558-561; The people such as Peterson, 2002, Bioconjugate Chem., 13,845-854; The people such as Erbacher, 1999, Journal of Gene Medicine Preprint, 1,1-18; The people such as Godbey, 1999, PNAS USA, 96,5177-5181; The people such as Godbey, 1999, Journal of Controlled Release, 60,149-160; The people such as Diebold, 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99,14640-14645; Sagara, U.S. Patent number 6,586,524 and U.S. Patent Application Publication No. 20030077829).

Nucleic acid molecule can be compound with rupture of membranes agent, such as in those rupture of membranes agent described in U.S. Patent Application Publication No. 20010007666.Described one or more rupture of membranes agent and nucleic acid molecule also can be compound with cation lipid or auxiliary lipid molecule, such as at U.S. Patent number 6,235, and those lipids described in 310.

Delivery system can comprise for example water-based and non-aqueous gel, paste, multiple emulsion, microemulsion, liposome, ointment, water-based and non-aqueous solution agent, lotion, aerosol, hydrocarbon bases and powder, and can comprise vehicle, for example, for example, such as solubilizing agent, penetration enhancers (lipid acid, fatty acid ester, fatty alcohol and amino acid) and hydrophilic polymer (polycarbophil and polyvinylpyrrolidone).In one embodiment, pharmaceutically acceptable carrier is liposome or dermal penetration enhancer.The limiting examples of the liposome that can use together with compound of the present invention comprise following these: (1) CellFectin, positively charged ion proton N, NI, NII, NIII-tetramethyl--N, NI, NII, 1:1.5 (M/M) Liposomal formulation (GIBCO BRL) of NIII-tetra-palmityl spermine and DOPE (DOPE); (2) Cytofectin GSV, 2:1 (M/M) Liposomal formulation (Glen Research) of cation lipid and DOPE; (3) DOTAP (N-[1-(the oily acyloxy of 2,3-bis-)-N, N, N-trimethylammonium-ammonium methyl-vitriol) (Boehringer Manheim); And (4) Lipofectamine, 3:1 (M/M) Liposomal formulation of polycationic lipid DOSPA, neutral lipid DOPE (GIBCO BRL) and dialkyl group amino acid (DiLA2).

Delivery system can comprise patch, tablet, suppository, hysterophore, gelifying agent, water-based and non-aqueous solution agent, lotion and creme, and can comprise vehicle, for example, for example, such as solubilizing agent and toughener (propylene glycol, biliary salts and amino acid) and other media thing (polyoxyethylene glycol, glycerine, fatty acid ester and derivative, and hydrophilic polymer, such as Vltra tears and hyaluronic acid).

Nucleic acid molecule can comprise biological conjugate, for example, as people such as Vargeese, and U.S. Patent number 10/427,160, U.S. Patent number 6,528,631, U.S. Patent number 6,335,434, U.S. Patent number 6,235,886, U.S. Patent number 6,153,737, U.S. Patent number 5,214,136, U.S. Patent number 5,138, the nucleic acid conjugates described in 045.

Composition disclosed herein, method and test kit can comprise expression vector, the nucleotide sequence that this expression vector comprises in the mode that allows nucleic acid molecule to express at least one nucleic acid molecule disclosed herein of encoding.The method of introducing cellular environment for nucleic acid molecule maybe being expressed to one or more carriers of dsRNA chain depends on the type of cell and the formation of environment thereof.Nucleic acid molecule or vector construction body can directly be introduced cell (, in cell); Or introduce cavity, clearance space in extracellular, and introduce organism circulation, per os is introduced, and maybe can introduce by organism or cell are placed in to the solution bath that contains dsRNA.Cell is preferably mammalian cell, is more preferably human cell.The nucleic acid molecule of expression vector can include adopted region and antisense region.Antisense region can comprise and the coding RNA of HES1, HES5, HEY2, CDKN1B or NOTCH1 or the sequence of DNA sequence dna complementation, and has adopted region can comprise and the sequence of antisense regional complementarity.Nucleic acid molecule can comprise two different chains with complementary sense and antisense region.Nucleic acid molecule can comprise the single chain with complementary sense and antisense region.

For example, can express from the transcriptional units that inserts DNA or RNA carrier with the nucleic acid molecule of the gene (HES1, HES5, HEY2, CDKN1B or NOTCH1mRNA, SEQ ID NO:1,2,10,7 or 11) of tone coded target RNA molecule under target RNA interaction of molecules also.Recombinant vectors can be DNA plasmid or virus vector.The nucleic acid molecule of expressing viral vector can based on but be not limited to adeno-associated virus, retrovirus, adenovirus or Alphavirus and build.Recombinant vectors that can express nucleic acid molecule can as described hereinly be sent, and stays in target cell.Or, can use the virus vector that allows nucleic acid molecule transient expression.Examples of such carriers can be used where necessary repeatedly.For example, once after expressing, nucleic acid molecule, in conjunction with also down-regulated gene function or expression, disturbs (RNAi) via RNA.Sending of nucleic acid molecule expression vector can be general, such as passing through intravenously or intramuscular administration, by topical application, by being administered to the in vitro target cell of experimenter and then introducing experimenter, or by allowing to introduce any other means in required target cell.

The nucleotide sequence that expression vector can comprise at least one nucleic acid molecule disclosed herein of encoding by the mode that allows nucleic acid molecule to express.For example, carrier can comprise the sequence that coding comprises two chains of the nucleic acid molecule of duplex.Carrier can also comprise coding oneself complementation also thereby form the single core acid molecule of nucleic acid molecule.The limiting examples of this type of expression vector is people such as Paul, 2002, Nature Biotechnology, 19,505; Miyagishi and Taira, 2002, Nature Biotechnology, 19,497; The people such as Lee, 2002, Nature Biotechnology, 19,500; And the people such as Novina, 2002, Nature Medicine, describes in online publishing doi:10.1038/nm725 in advance to some extent.Expression vector for example also can be included in, in Mammals (mankind) cell.

One or two chains of expression vector codified nucleic acid duplex, or oneself is hybridized to the wall scroll oneself complementary strand of nucleic acid duplex.The nucleotide sequence of coding nucleic acid molecule can be operably connected (referring to the people such as such as Paul, 2002, Nature Biotechnology, 19,505 by the mode that allows nucleic acid molecule to express; Miyagishi and Taira, 2002, Nature Biotechnology, 19,497; The people such as Lee, 2002, Nature Biotechnology, 19,500; And the people such as Novina, 2002, Nature Medicine, in advance online publishing doi:10.1038/nm725).

Expression vector can comprise following one or more: a) transcription initiation region (for example eucaryon polI, II or III initiation region); B) Transcription Termination region (for example eucaryon pol I, II or III stop region); C) intron and d) nucleotide sequence of at least one in coding nucleic acid molecule, wherein said sequence is to allow nucleic acid molecule to express and/or the mode of sending is operably connected to initiation region and stops region.Carrier optionally comprises the albumen open reading frame (ORF) in 5 ' side or the 3 ' side of the sequence that is operatively coupled on coding nucleic acid molecule; And/or intron (intervening sequences).

Transcribing of sequence of nucleic acid molecules can be by the promoters driven of eucaryotic RNA polysaccharase I (pol I), rna plymerase ii (pol II) or rna plymerase iii (pol III).From the transcript of pol II or pol III promotor all cells all with high level expression; Near the character of the gene regulating sequence (enhanser, silencer etc.) that the expression of given pol II promotor in given cell type exists depending on.Also can use prokaryotic rna polymerase promotor, prerequisite is that prokaryotic rna polymerase is at suitable cells (Elroy-Stein and Moss, 1990, Proc.Natl.Acad.Sci.USA, 87,6743-7; Gao and Huang 1993, Nucleic Acids Res., 21,2867-72; The people such as Lieber, 1993, Methods Enzymol., 217,47-66; The people such as Zhou, 1990, Mol.Cell.Biol., 10,4529-37).Several researchists are verified such as, by the nucleic acid molecule of this class promoter expression can play a role in mammalian cell (people such as Kashani-Sabet, 1992, Antisense Res.Dev., 2,3-15; The people such as Ojwang, 1992, Proc.Natl.Acad.Sci.USA, 89,10802-6; The people such as Chen, 1992, Nucleic Acids Res., 20,4581-9; The people such as Yu, 1993, Proc.Natl.Acad.Sci.USA, 90,6340-4; The people such as L ' Huillier, 1992, EMBO J., 11,4411-8; The people such as Lisziewicz, 1993, Proc.Natl.Acad.Sci.U.S.A, 90,8000-4; The people such as Thompson, 1995, Nucleic Acids Res., 23,2259; Sullenger & Cech, 1993, Science, 262,1566).More particularly, transcriptional units such as the gene derived from coding U6 small nut (snRNA), transfer RNA (tRNA) and adenovirus VA RNA is used in the required RNA molecule that generates high density in cell, such as siNA, (people such as Thompson, sees above; Couture and Stinchcomb, 1996, see above; The people such as Noonberg, 1994, Nucleic Acid Res., 22,2830; The people such as Noonberg, U.S. Patent number 5,624,803; The people such as Good, 1997, Gene Ther., 4,45; The people such as Beigelman, International PCT publication No. WO96/18736).Above transcribed nucleic acid unit can be incorporated in variety carrier to introduce mammalian cell, include but not limited to plasmid DNA carrier, viral DNA carrier (such as adenovirus or gland relevant viral vector) or viral rna vector (such as retrovirus or Alphavirus carrier) (referring to Couture and Stinchcomb, 1996 see above).

Nucleic acid molecule can be expressed (for example Izant and Weintraub, 1985, Science, 229,345 from eukaryotic promoter in cell; McGarry and Lindquist, 1986, Proc.Natl.Acad.Sci., USA 83,399; The people such as Scanlon, 1991, Proc.Natl.Acad.Sci.USA, 88,10591-5; The people such as Kashani-Sabet, 1992, Antisense Res.Dev., 2,3-15; The people such as Dropulic, 1992, J.Virol., 66,1432-41; The people such as Weerasinghe, 1991, J.Virol., 65,5531-4; The people such as Ojwang, 1992, Proc.Natl.Acad.Sci.USA, 89,10802-6; The people such as Chen, 1992, Nucleic Acids Res., 20,4581-9; The people such as Sarver, 1990Science, 247,1222-1225; The people such as Thompson, 1995, Nucleic Acids Res., 23,2259; The people such as Good, 1997, Gene Therapy, 4,45).Those skilled in the art will recognize that, any nucleic acid all can be by suitable DNA/RNA vector expression in eukaryotic cell.The activity of this type of nucleic acid can strengthen (people such as Draper, PCT WO 93/23569, and the people such as Sullivan, PCT WO94/02595 from the originally release of transcript by it under the effect of enzymatic nucleic acid; The people such as Ohkawa, 1992, Nucleic Acids Symp.Ser., 27,15-6; The people such as Taira, 1991, Nucleic Acids Res., 19,5125-30; The people such as Ventura, 1993, Nucleic Acids Res., 21,3249-55; The people such as Chowrira, 1994, J.Biol.Chem., 269,25856).

The virus formulation body being packaged in virus particle is efficiently introduced expression construct in cell and is transcribed the dsRNA construct of being encoded by this expression construct realizing.

Per os introducing method comprises RNA is directly mixed with the food of organism, and engineered method, wherein by as the material of food through engineered with expressed rna, then offer affected organism.Can adopt physical method that nucleic acid molecule solution is introduced to cell.The physical method of introducing nucleic acid comprises the solution that injection contains nucleic acid molecule, bombards by the particle being covered by nucleic acid molecule, and cell or organism are immersed to the solution of RNA, or in the situation that there is nucleic acid molecule, carries out the electroporation of cytolemma.In one embodiment, provide the cell that comprises nucleic acid molecule disclosed herein herein.

Can use known in the art for nucleic acid being introduced to the additive method of cell, such as the transhipment of chemistry mediation, such as calcium phosphate etc.Therefore, can introduce nucleic acid molecule together with one or more the component of carrying out in following activity: strengthen the absorption of cell to RNA, the annealing of promotion duplex chain, chain or the otherwise inhibition/downward of intensifier target gene of stabilizing annealing.

Polymer nanocomposite capsule or microballoon are conducive to seal or the dsRNA of combination transports and be discharged into cell.They comprise polymkeric substance and monomer material, especially comprise PBCA.The general introduction (referring to Kreuter, 1991) of material and manufacture method is disclosed.Generating in polyreaction/nanoparticle the polymer materials itself being formed by monomer and/or oligopolymer precursor in step is known in the prior art, with technician in nanoparticle manufacture field can according to common skill, in addition suitably the molecular weight and molecualr weight distribution of the polymer materials of selection be the same.

Nucleic acid molecule can be mixed with to microemulsion.Microemulsion is the system of water, oil and amphiphile, its be single optically isotropy and in thermokinetics stable liquor.Conventionally, microemulsion is prepared in the following manner: first oil content is dispersed in aqueous tenside solution, then adds the 4th kind of enough component, be generally the alcohol of medium chain degree to form transparent system.

The tensio-active agent that can be used for preparing microemulsion includes but not limited to ionic surface active agent independent or that combine with cosurfactant, nonionogenic tenside, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid ester, four glyceryl monolaurates (ML310), four glyceryl monooleates (MO310), six glyceryl monooleates (PO310), six glycerine five oleic acid esters (PO500), ten monocaprins (MCA750), SY-Glyster MO 750 (MO750), ten glycerine sesquioleate (SO750), SY-Glyster DAO 750 (DA0750).Cosurfactant is generally short chain alcohol such as ethanol, 1-propyl alcohol and n-butyl alcohol and plays in the following manner the effect that increases interface motion: infiltrate Surfactant Films, and therefore because the void space producing among surfactant molecule forms disordered film.

Delivery formulation can comprise water-soluble degradable cross-linked polymer, this polymkeric substance comprises one or more degradable crosslinked lipid parts, one or more PEI part and/or one or more mPEG (methyl ether derivative of PEG, i.e. methoxyl group PEG).

dosage

Useful dosage to be administered and specific mode of administration change basis such as the factor of cell type, or for using in body, change according to following aspect: therapeutic or diagnostic purposes and the formulation of age, body weight and specific experimenter and region to be treated thereof, specific nucleic acid used and delivering method, imagination, for example suspension agent, emulsion, micella or liposome, as will be apparent to those skilled in the art.Conventionally, with lower horizontal application dosage, and increase until realize required effect.

Therefore,, for this paper object, " treatment effective dose " is by this type of is considered and determines as known in the art.Dosage must effectively be realized improvement, includes but not limited to improve survival rate or recovers sooner or improve or eliminate symptom and those skilled in the art and be chosen as other indexs of appropriate action.

Can introduce appropriate inhibitor, for example nucleic acid molecule, and this tittle can use standard method to determine from experience.In cellular environment, the effective concentration of each nucleic acid molecule material can be approximately 1 and flies mole, approximately 50 fly mole, 100 fly mole, 1 picomole, 1.5 picomole, 2.5 picomole, 5 picomole, 10 picomole, 25 picomole, 50 picomole, 100 picomole, 500 picomole, 1 nmole, 2.5 nmole, 5 nmoles, 10 nmoles, 25 nmoles, 50 nmoles, 100 nmoles, 500 nmoles, 1 micromole, 2.5 micromole, 5 micromoles, 10 micromoles, 100 micromoles or higher.

In general, for the active dose of the mankind's nucleic acid compound single dose scheme every day 1ng/kg to about 20-100 mg/kg (mg/kg) recipient body weight, preferably every day, about 0.01mg was to the scope of about 2-10mg/kg recipient body weight, every day twice of potion or every day or three times or more times, continue 1 to 4 week or longer cycle.The suitable dose unit of nucleic acid molecule can be in the scope of 0.001 to 0.25 milligram of every kilogram of recipient's body weight every day, or in the scope of every kg body weight 0.01 to 20 microgram every day, or in the scope of every kg body weight 0.01 to 10 microgram every day or in the scope of every kg body weight 0.10 to 5 microgram every day or in the scope of every kg body weight 0.1 to 2.5 microgram every day.Dosage can be the every kg body weight of 0.01ug to 1g (for example every kg body weight of 0.1ug, 0.25ug, 0.5ug, 0.75ug, 1ug, 2.5ug, 5ug, 10ug, 25ug, 50ug, 100ug, 250ug, 500ug, 1mg, 2.5mg, 5mg, 10mg, 25mg, 50mg, 100mg, 250mg or 500mg).

About 0.1mg can be used for treating above indication (about 0.5mg to every of about 7g experimenter every day) to the dosage level of the every kg body weight of about 140mg every day.Can be combined with carrier substance and change according to the host for the treatment of and specific mode of administration with the amount of the activeconstituents that forms single dose form.Dosage unit form comprises about 1mg conventionally to about 500mg activeconstituents.

Be to be understood that, the concrete dosage level of any particular subject depends on many factors, comprises activity, age, body weight, general health situation, sex, diet, time of application, route of administration, excretion rate, the drug regimen of particular compound used and the seriousness of the specified disease of receiving treatment.

The pharmaceutical composition that comprises inhibitor disclosed herein (for example nucleic acid molecule) can use once according to needs medically, once a day (QD), one day twice (bid), one day three times (tid), one day four times (qid), with any interval and continue to continue any time any time or by applying continuously.Therapeutical agent also can be by being included in two, three, four, five, six or the dosage device administration of more sub-doses of using with suitable interval in one day.In the case, the nucleic acid molecule being included in each sub-doses can be correspondingly less, to realize total dosage device every day.Also dosage device can be combined into the single dose through a couple of days, for example, use conventional extended release preparation, it provides and continues to discharge with consistent dsRNA within the time of a couple of days.Extended release preparation is known in the art.Dosage device can comprise corresponding multiple every per daily doses.Composite composition by this way, makes the summation of the multiple unit of nucleic acid comprise together enough dosage.Can be exposed to infringement (, ear toxin, physical abuse etc.) before, during or the combination of administering therapeutic agent afterwards.

pharmaceutical composition, test kit and container

The present invention also provides composition, combination, commercial package, test kit, container and preparation, the inhibitor that it comprises the expression of lowering as herein provided HES1, HES5, HEY2, CDKN1B or NOTCH1, for example nucleic acid molecule (for example siNA molecule), to use to patient or to distribute nucleic acid molecule.Test kit can comprise at least one container and at least one label.Suitable container comprises for example bottle, cillin bottle, syringe and test tube.Container can be formed by multiple material such as glass, metal or plastics.Container can hold aminoacid sequence, small molecules, nucleotide sequence, cell mass and/or antibody and/or realize any other required component of related experiment, prognosis, diagnosis, prevention and therapeutic purpose.The explanation of indication and/or this type of purposes can be included on this type of container or together with this type of container, the same with other compositions or instrument with the reagent for these objects.

Alternatively, container can hold the composition of effective treatment, diagnosis, prognosis or prevention illness, and can there is sterile access port (for example, container can be parenteral solutions bag or has the cillin bottle of the plug that can pierce through by hypodermic needle).Promoting agent in composition can be can specific binding HES1, HES5, HEY2, CDKN1B or NOTCH1mRNA and/or lower the nucleic acid molecule of HES1, HES5, HEY2, CDKN1B or NOTCH1 function.

Test kit can further comprise second container, and it holds pharmaceutically acceptable damping fluid, such as phosphate buffered saline (PBS), Ringer's solution and/or glucose solution.It can further comprise from business and the desirable other materials of user perspective, comprises other damping fluids, thinner, strainer, agitator, syringe needle, syringe and/or has the package insert that adapts to this and/or operation instruction.

Federal law requires pharmaceutical composition must obtain the approval of federation or national governmental agencies for human treatment.In the U.S., to implement to be responsible for by food and medicine Surveillance Authority, it has issued the suitable specification that obtains this approval, refers to the 301-392 in 21U.S.C. §.Most of foreign countries/area needs similar approval.Specification is different between country variant/area, but each program knows for those skilled in the art, and composition provided in this article, combination and method be corresponding these specifications that meets preferably.

Combination disclosed herein can combine and be used for the treatment of disease, illness or the obstacle relevant with the expression of HES1, HES5 and HEY2 or CDKN1B, NOTCH1 and HEY2 individually or to other therapies, such as disease, damage, illness or pathology in ear, vestibular sense system, and to HES1, HES5 and HEY2 or CDKN1B, NOTCH1 are relevant with the expression level of HEY2 in cell or tissue or by any other disease or illness in response to described level.Therefore, composition disclosed herein, combination, commercial package, test kit and method can comprise packaging nucleic acid molecule disclosed herein, and it comprises label or specification sheets.Label can comprise the operation instruction of nucleic acid molecule, such as being used for the treatment of or disease, obstacle, damage and the illness of preventing ear or vestibular system, include but not limited to Meniere, sense of hearing wound, deafness, hearing loss, presbyacusis and any other disease disclosed herein or illness.Label can comprise the operation instruction of nucleic acid molecule, such as being used for the treatment of or preventing or alleviate neuronal degeneration.Neuronal degeneration comprises the sex change of for example auditory nerve (being also responsible for sound and balancing information to be delivered to brain from inner ear also referred to as vestibulocochlear nerve or auditory nerve); Communicate information to the modification of the hair cell of the inner ear of brain via auditory nerve, described auditory nerve is made up of cochlea nerve and vestibular nerve, and occurs and enter encephalic by the internal auditory meatus temporal bone (or interior auditory canal) together with facial nerve from oblongata.Label can comprise the operation instruction of nucleic acid molecule, such as individually or combine with other therapies and be used for the treatment of or prevent to relate to or by any other disease or illness in response to HES1, HES5 in cell or tissue and HEY2 expression level or CDKN1B, NOTCH1 and HEY2 expression level.Label can comprise the operation instruction that reduces and/or lower the expression of HES1, HES5 and HEY2 or CDKN1B, NOTCH1 and HEY2." specification sheets " is used in reference to the explanation in the commercial package that is generally comprised within therapeutic product, and it comprises about indication, consumption, dosage, usage, contraindication, by the other treatment product of being combined with wrapped product and/or about the information of warning etc. that uses this type of therapeutic product.

Person of skill in the art will appreciate that, other treatment known in the art, medicine and therapy can easily for example, combine with nucleic acid molecule (dsNA molecule) herein, and therefore take into account herein.

methods for the treatment of

On the other hand, the present invention relates to the experimenter's who is used for the treatment of needs treatment disease or the obstacle relevant to HES1, HES5, HEY2, CDKN1B or NOTCH1 unconventionality expression, comprise to experimenter and use a certain amount of reduction or suppress the inhibitor of HES1, HES5 and HEY2 genetic expression or the inhibitor of reduction or inhibition CDKN1B, NOTCH1 and HEY2 genetic expression.

In one embodiment, can be by nucleic acid molecule for lowering or suppressing HES1, HES5, HEY2, CDKN1B or NOTCH1 and/or the expression of HES1, the HES5, HEY2, CDKN1B or the NOTCH1 albumen that are produced by HES1, HES5, HEY2, CDKN1B or NOTCH1 and/or the haplotype polymorphism for example, to disease or illness (neurodegeneration) relevant.The analysis of HES1, HES5, HEY2, CDKN1B gene and/or albumen or rna level be can be used for to qualification and there are the experimenter of this type of polymorphism or those experimenters in there is speciality as herein described, illness or disease risks.These experimenters for example can accept following treatment: treat with nucleic acid molecule disclosed herein and any other composition of can be used for the disease that treatment is relevant to HES1, HES5, HEY2, CDKN1B or NOTCH1 genetic expression.Therefore, the analysis of HES1, HES5, HEY2, CDKN1B or NOTCH1 gene and/or albumen or rna level be can be used for determining treatment type and the therapeutic process in the time that treatment experimenter treats.Monitoring albumen or rna level can be used for predicted treatment result, and some gene of definite adjusting and speciality, illness or disease-related and/or the level of albumen and/or the combination of activity and the effect of composition.

Provide herein the expression of the target gene that is selected from the gene that is transcribed into the mRNA of SEQ ID NO:1,2,10,7 or 11 shown in any one is suppressed at least 40%, preferably 50%, 60% or 70%, more preferably 75%, 80% or 90% method compared with the control, comprise the mRNA transcript of target gene disclosed herein is contacted with combination provided in this article or composition.

In one embodiment, oligoribonucleotide suppresses one or more in target gene disclosed herein, therefore suppresses to be selected from inhibition, the inhibition of polypeptide and the inhibition of mrna expression of gene function.

In one embodiment, compound suppresses target polypeptide, therefore suppresses to be selected from inhibition (it is for example especially measured and study by enzymatic determination or with the combination of known action of natural gene/polypeptide), the inhibition (it is for example especially studied by Western trace, ELISA or immunoprecipitation) of albumen and the inhibition (it is for example especially studied by Northern trace, quantitative RT-PCR, in situ hybridization or microarray hybridization) of mrna expression of function.

In one embodiment, compound is lowered Mammals polypeptide, therefore suppresses to be selected from downward (it is for example especially measured and study by enzymatic determination or with the combination of known action of natural gene/polypeptide), the downward (it is for example especially studied by Western trace, ELISA or immunoprecipitation) of albumen and the downward (it is for example especially studied by Northern trace, quantitative RT-PCR, in situ hybridization or microarray hybridization) of mrna expression of function.

In other embodiments, provide treatment to suffer from the patient's of following disease method herein, this disease raises with the level of the mammalian genes that is selected from the gene that is transcribed into the mRNA of SEQ ID NO:1,2,10,7 or 11 shown in any one, the method comprises to combination as disclosed herein or the composition of patient's administering therapeutic effective dose, thereby treats described patient.

The method, combination and the composition that suppress mammalian genes as disclosed herein or polypeptide discuss in detail herein, and any described molecule and/or composition are all used for the treatment of the patient who suffers from any described illness valuably.Use the new treatment of known compound and composition to fall within the scope of the invention.

In multiple embodiments, method disclosed herein comprises the compound of the expression of the downward hearing loss genes involved of administering therapeutic significant quantity.So-called " being exposed to toxic agents " refers to and toxic agents is exposed to or contacts Mammals.Toxic agents can have toxicity to neural system.Being exposed to toxic agents can be by directly using and occurs, for example for example, occur by absorbing or use food, medicine or therapeutical agent (chemotherapeutics), occur by accidental pollution, or for example, occur by environmental exposure (air or water expose).

The method of prevention auditory nerve (vestibulocochlear nerve or auditory nerve) sex change is also provided herein, and described nerve is responsible for transmitting from inner ear to brain sound and balancing information.The hair cell of inner ear is by auditory nerve to brain transmission of information, and auditory nerve is made up of cochlea nerve and vestibular nerve, and occurs and enter encephalic by the internal auditory meatus temporal bone (or interior auditory canal) together with facial nerve from oblongata.

The technique that pharmaceutical compositions is also provided herein, it comprises:

One or more duplex molecules disclosed herein are provided; And

By described molecule and pharmaceutically acceptable carrier compounding.

In a preferred embodiment, with pharmacy effective dose by the molecule for the preparation of pharmaceutical composition and carrier compounding.In specific embodiment, compound of the present invention is coupled to the such as cholesterol of molecule that steroid or lipid or another are suitable.

Nucleic acid molecule disclosed herein can be lowered in sequence-specific mode the expression of HES1, HES5, HEY2, CDKN1B or NOTCH1.Nucleic acid molecule can comprise sense strand and antisense strand, and it comprises at least in part the continuous nucleotide with the part complementation (antisense) of HES1, HES5, HEY2, CDKN1B or NOTCH1mRNA.

In some embodiments, the specific dsRNA of HES1, HES5, HEY2, CDKN1B or NOTCH1 can be combined with other treatment agent and/or the specific dsRNA of other molecular targets (such as, but not limited to various short apoptogenes).

Be used for the treatment of or prevent the method for HES1, HES5, HEY2, CDKN1B or NOTCH1 relative disease or illness in experimenter or organism to comprise experimenter or organism are contacted under the condition that is suitable for lowering the expression of described gene in described experimenter or organism with combination provided in this article or composition.

Be used for the treatment of or prevent the method for the ear's obstacle in experimenter or organism to comprise experimenter or organism are contacted under the condition that is suitable for lowering HES1, HES5, HEY2, CDKN1B or the expression of NOTCH1 gene in described experimenter or organism with combination provided in this article or composition.

In preferred embodiments, the experimenter who treats is warm-blooded animal and is especially the Mammals that comprises people.

Method disclosed herein comprises treating effective dose and uses combination or the composition of inhibition compound to experimenter, to treat described experimenter, described compound is lowered the expression of HES1, HES5 and HEY2 or CDKN1B, NOTCH1 and HEY2.

Lower HES1, HES5, HEY2, CDKN1B or NOTCH1 be HES1 inhibitor or its pharmacy acceptable salt or prodrug especially, the combination of HES5 inhibitor or its pharmacy acceptable salt or prodrug and HEY2 inhibitor or its pharmacy acceptable salt or prodrug or CDKN1B inhibitor or its pharmacy acceptable salt or prodrug, the method of the combination of NOTCH1 inhibitor or its pharmacy acceptable salt or prodrug and HEY2 inhibitor or its pharmacy acceptable salt or prodrug, combination and composition have been described in detail herein, and any described combination and/or composition all can be used for the treatment of the experimenter who suffers from any described illness valuably.The sense strand and the antisense strand oligonucleotide sequence that can be used for generating dsRNA illustrate herein.Can be used for the preferred oligonucleotide sequence of the dsRNA for preparing the expression of lowering HES1 shown in SEQ ID NO:26667-26690 and 26691-26706; Can be used for the preferred oligonucleotide sequence of the dsRNA for preparing the expression of lowering HES5 shown in SEQ ID NO:26707-26724 and 26725-26732; Can be used for the preferred oligonucleotide sequence of the dsRNA for preparing the expression of lowering HEY2 shown in SEQ ID NO:26779-26784 and 26785-26788; Can be used for the preferred oligonucleotide sequence of the dsRNA for preparing the expression of lowering CDKN1B shown in SEQ ID NO:26867-26886 and 26887-26900; Or the preferred oligonucleotide sequence that can be used for the dsRNA for preparing the expression of lowering NOTCH1 is shown in SEQ ID NO:26901-26910 and 26911-26912.

Method disclosed herein comprises treating effective dose uses composition as disclosed herein or the combination of inhibition compound (lowering the expression of the gene shown in SEQ ID NO:1,2,10,7 or 11) to experimenter, especially the composition of oligonucleotide compound or combination, to treat described experimenter.

" treatment " refers to therapeutic treatment and preventative or control property measure, wherein object is prevention obstacle or the symptom that alleviates obstacle, such as dysaudia or damage (or disequilibrium), the diseases related relevant necrocytosis of hearing loss that prevention or minimizing and this paper are listed, promotes ear (sensation) cell regeneration or promotes the sustenticular cell in inner ear to be divided into ear (sensation) cell.Need those experimenters for the treatment of to comprise to suffer from disease or illness those, have occur disease or illness tendency those and want preventing disease or those of illness.Composition disclosed herein or be combined in disease or illness occur before, during or use afterwards.

Be not bound by theory, hearing loss can be because of due to apoptosis inner ear hair cells infringement or loss, and wherein this infringement or loss are caused by infection, physical abuse, ototoxicity loud, that aging or especially chemical cause.Ear toxin comprises curative drug, comprise antineoplastic agent, salicylate, quinine and aminoglycoside antibiotics, the pollutent in food or medicine, and environment or industrial pollutants.Conventionally, treat to prevent or alleviate ototoxicity, especially because of or estimate the ototoxicity causing because of administering therapeutic medicine.Preferably, after exposure, treat immediately effective composition to prevent or to alleviate ototoxicity effect.More preferably, by before using ototoxic drug or being exposed to ear toxin or use concomitantly and treatment is prophylactically provided.

The hearing loss relevant to the disclosure can be because relating to due to the end-organ pathology of inner ear hair cells, for example sense of hearing wound, viral endolymphatic labyrinth inflammation, Meniere.Hearing loss comprises tinnitus, and it is the perception to sound in the situation that not existing sound to stimulate, and can be intermittent or successional, and wherein diagnosis has phonosensitive dysacousis.Hearing loss can be because of due to bacterium or virus infection, such as in zoster oticus; The suppurative labyrinthitis being caused by acute otitis media, purulent meningitis, chronic otitis media, sudden deafness (comprising viral origin), the viral endolymphatic labyrinth inflammation for example being caused by virus, comprises parotitis, measles, influenza, varicella, mononucleosis and adenovirus.Hearing loss can be geneogenous, such as by rubella, when birth anoxic, cause into inner ear, erythroblastosis fetalis and heredity illness (Waardenburg's syndrome and gargoylism) because of bleeding of using to parent that wound during ototoxic drug causes.

Hearing loss can be that noise causes, conventionally owing to damaging the noise that is greater than 85 decibels (db) of inner ear.One specific aspect, hearing loss is caused by the sense of hearing part that the affects inner ear especially ototoxic drug of inner ear hair cells.Be incorporated herein by reference The Merck Manual of Diagnosis and Therapy, the 14th edition, (1982), Merck Sharp & Dome Research Laboratories, N.J. the 196th, 197,198 and 199 chapters, and corresponding chapters and sections in nearest the 16th edition, comprise the 207th and 210 chapters of the description and the diagnosis that relate to hearing and disequilibrium.

In one embodiment, the invention provides and be used for the treatment of (or balance) obstacle of suffering from hearing or have the Mammals of this tendency or in gene inhibition of the present invention mammiferous method of prophylactic treatment be useful in the situation that.The method will prevent or alleviate generation or the seriousness of hearing (balance) obstacle, and described obstacle will cause because of inner ear cell injury, loss or sex change, especially because ototoxicity agent causes.In some embodiments, the method comprises HES1 inhibitor, HES5 inhibitor and the HEY2 inhibitor of administering therapeutic significant quantity.In other embodiments, the method comprises CDKN1B inhibitor, NOTCH1 inhibitor and the HEY2 inhibitor of administering therapeutic significant quantity.

Target of the present disclosure is to be provided for treating Mammals to prevent, to alleviate or to treat method, combination and the composition of hearing loss, obstacle or the unbalance hearing illness optionally being caused by ear toxin by the administration composition disclosed herein to needs treatment or combination.In some embodiments, the method is used for the treatment of dysaudia or damage, and wherein ototoxicity causes because of the ototoxic drug of administering therapeutic significant quantity.Typical ototoxic drug is chemotherapeutics, for example antineoplastic agent, and microbiotic.Other possible material standed fors comprise loop diuretic, quinine or quinine compounds and salicylate or salicylate compounds.

In some embodiments, combination provided in this article and composition and ear toxin are used jointly.For example, provide by using aminoglycoside antibiotics and treated improving one's methods of mammalian infections, improvement comprises to the composition or the combination that this treatment are had to downward HES1, the HES5 of patient's administering therapeutic significant quantity of needs and the expression of HEY2 or lower the inhibitor (especially dsRNA) of the expression of CDKN1B, NOTCH1 and HEY2, to alleviate or to prevent the hearing loss that the ear toxin relevant to microbiotic causes.Lower for example preferably topical application in inner ear of dsRNA of compound of the expression of HES1, HES5, HEY2, CDKN1B or NOTCH1.

In another embodiment, provide by using chemotherapy compound and treated improving one's methods of mammalian cancer, wherein improve composition as disclosed herein or the combination of patient's administering therapeutic significant quantity comprising to this treatment being had to needs, to alleviate or to prevent the hearing loss that the ear toxin relevant to chemotherapeutics causes.Alleviate or prevent the composition of the hearing loss that ear toxin causes or combination and for example comprise especially as disclosed herein the composition of dsRNA molecule and combinatorial optimization and be applied directly to cochlea as the naked dsRNA in the vehicle such as PBS or other physiological solutions, but can use by delivery vehicle as above alternatively.

In another embodiment, methods for the treatment of is applicable to the hearing loss that treatment causes because using chemotherapeutics, to treat its ototoxicity side effect.

In another embodiment, described method, composition and combination are applicable to be generally used for treating the quinine of malaria and the hearing loss that synthetic substitute causes thereof because using, to treat its ototoxicity side effect.

In some embodiments of combination provided in this article, by using two or three inhibitor (being dsRNA), wherein each prepares separately and uses and realize combination treatment, or realizes by use described inhibitor in unitary agent.Combination treatment is also contained other combinations.For example, can be by formulated together two kinds of inhibitor, and co-administered with the independent preparation that comprises the 3rd inhibitor.Although can side by side use two or more preparations in combination treatment, needn't be like this.For example, use the first inhibitor (or combination of inhibitor) and can use the second inhibitor (or combination of inhibitor) several minutes before, a few hours, a couple of days or several weeks.Therefore, described two or more inhibitor can be in each other several minutes or each other one to a few hours or using to a couple of days or within several weeks each other each other.In some cases, even longer interval is also possible.Described two or more inhibitor for combination treatment can or can be present in patient body when difference.Combination treatment comprise for the inhibitor of this combination one or more twice or more times use.For example, if dsRNA1 and dsRNA2 are (, wherein dsRNA1 target gene 1 and dsRNA2 target gene 2) to be used in combination, can they be used one or more time in order by any combination, for example, in the following order: dsRNA1-dsRNA2, dsRNA2-dsRNA1, dsRNA1-dsRNA2-dsRNA1, dsRNA2-dsRNA1-dsRNA2, dsRNA1-dsRNA1-dsRNA2, dsRNA1-dsRNA2-dsRNA2 etc.

The form that combination can single medicine preparation is as disclosed herein used, optionally together with pharmaceutically acceptable diluent or carrier.Being called each component of such combination of inhibitor can be side by side, concomitantly, individually or according to priority be used by identical or independent pharmaceutical preparation.

In some embodiments, every kind of inhibitor by identical approach by identical or used by different pharmaceutical compositions.In other embodiments, for the first inhibitor, the second inhibitor and the 3rd inhibitor, to use identical route of administration be impossible or be not preferred.Skilled in the art will recognize that independent or with the optimal application pattern of every kind of inhibitor of array configuration.

As used herein, mean in time period that being applied in of the second inhibitor be no more than approximately 5 minutes after the using of the first inhibitor about the term " substantially side by side " of using at least two kinds of inhibitor, preferably within the time period of approximately 1 minute, more preferably within the time period of approximately 30 seconds, and most preferably side by side use by identical or independent pharmaceutical preparation with the first inhibitor.Similarly, about using of three kinds of inhibitor, using in the time period that can be no more than approximately 5 minutes after the using of the second inhibitor of the 3rd inhibitor, preferably within the time period of approximately 1 minute, more preferably within the time period of approximately 30 seconds, and most preferably side by side use by identical or independent pharmaceutical preparation with the second inhibitor.

hearing regeneration

Sensation progenitor cell can develop into hair cell or sustenticular cell.Rejecting (ablation) research shows to remove hair cell and has changed the destiny of peripheral cell from sustenticular cell to hair cell.This response support is drawn a conclusion: hair cell generates and prevents that flanking cell from developing into the Inhibitory signal of hair cell.Such interaction is consistent with the lateral inhibition effect of Notch mediation.Hypothesis is consistent therewith, and two kinds of Notch part Jag2 and delta 1 (Dll1) raise fast in Atoh1 positive cell subgroup.The expression of these parts causes the activation of Notch 1 and two kinds of Notch path target gene HES1 and HES5 to transcribe increase in the cell that will develop into sustenticular cell.In this path, the disappearance of any gene causes excessively producing hair cell, thereby shows that Notch signal transduction is acting on progenitor cell from bringing into play aspect the transfer of hair cell destiny.This transfer mechanism is studied with the cell in Kolliker organ.First, Kolliker organ cell is enough to suppress hair cell with Atoh1 and HES1 cotransfection and forms, thereby show that it is the target of HES1 in ear that Atoh1 transcribes.Secondly, in Kolliker organ cell diaphragm the instantaneous activation of ATOH1 cause those cells in the activation of Notch signal transduction, and cause the inhibition of ATOH1 and hair cell destiny in the subgroup of those cells.

The people such as Zine (J Neurosci.200121 (13): 4712-20) have confirmed that progenitor cell is shaped into respectively IHC and OHC destiny has great importance for suppressing for HES1 and HES5 activity, and possible mechanism is antagonism Math1.This negative regulator is most important for producing the hair cell of correct number and inlaying (cochlear mosaic) for the normal cochlea of setting up single file IHC and three row OHC.In vestibular system, HES1 and HES5 also serve as negative regulator of hair cell differentiation in Utriculus and sacculus epithelial cell.Likely, lower the replacement that HES1 in cochlea and HES5 can be used for stimulating the auditory hair cell losing simultaneously.This type of research has obvious therapeutic value, is hearing loss and/or deaf common cause because lose auditory hair cell because of disease, wound and aging.

The details that compound disclosed herein can be used as some indication of therapeutical agent are therein described herein.

Aspect provided in this article and embodiment are described with exemplary approach, and should be understood that, term used is intended to have illustrative and non-limiting part of speech.

According to above-mentioned instruction, many modifications and variations are all possible.Therefore, should be understood that, within the scope of the appended claims, the present invention can put into practice by the mode outside specifically describing.

In entire chapter patent application, every kind of patent is announced (comprising United States Patent (USP)) and is quoted in the mode of author and time and the patent No..The disclosure entirety of these patent announcements and patent and patent application is incorporated to the application with way of reference, to more fully describe the prior art under the present invention.

The disclosure will below be shown specifically in conjunction with the embodiments, but should not be considered limited to this.

Quote any document herein and do not mean that and admit that this document suits prior art, or there is the patentability of any claim of the disclosure depending on material.Based on applicant's obtainable information in the time submitting to, do not form admitting this explanation exactness about any explanation on any literature content or date.

Embodiment

Without further elaborating, it is believed that those skilled in the art uses above stated specification can farthest utilize the present invention.Therefore, below preferred specific embodiments only should be understood to as exemplary, instead of restriction is subject to the invention of claims protection by any way.

Not specifically described standard molecular biology scheme known in the art is generally substantially with reference to Publication about Document herein: the people such as Sambrook, Molecular cloning:A laboratory manual, Cold Springs Harbor Laboratory, New-York (1989,1992); With the people such as Ausubel, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988); With the people such as Ausubel, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); And Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); With the people such as Watson, Recombinant DNA, Scientific American Books, New York; With people (editor) the Genome Analysis:A Laboratory Manual Series such as Birren, 1-4 volume, Cold Spring Harbor Laboratory Press, New York (1998); And as U.S. Patent number 4,666,828,4,683,202,4,801,531,5,192,659 and 5,272, the method being incorporated to shown in 057 and with way of reference.Polymerase chain reaction (PCR) carries out according to Standard PC R scheme: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990).Can be used for detecting the cell (people such as Testoni, Blood 1996, the 87:3822 that contain specific DNA and mRNA sequence in conjunction with the original position PCR of flow cytometry (FACS).) to carry out the method for RT-PCR be also well known in the art.

the vitro test of embodiment 1:dsRNA molecule

With the about 1.5-2x10 in every hole ⁵individual density (is HeLa cell and/or 293T cell for the siRNA of targeted human genoid by test cell; And be NRK52 cell (normal rat renal proximal tubule cell) and/or NMuMG cell (mammary gland of mouse epithelial cell line) for the siRNA of target rat/mouse gene) be inoculated into (70-80% degree of converging) in 6 orifice plates.

After 24 hours, by cell Lipofectamine ^tM2000 reagent (Invitrogen) carry out transfection with the concentration of 5nM or 20nM by dsRNA molecule.By cell at 37 DEG C at CO ₂incubation 72h in incubator.

As the positive control of transfection, use the dsRNA molecule of PTEN-Cy3 mark.Negative control by GFP dsRNA molecule as siRNA activity.After transfection when 72h, harvested cell, and from cell, extract RNA.Test transfection efficiency by fluorescence microscopy.Expressing in the cell of native gene, by the qPCR analytical procedure of target gene, measure and use the genetic expression of certain preferred siRNA structure generation to suppress per-cent.

body fluid/cell stability is measured

Tested as follows modified compound disclosed herein in the mankind, rat or mice plasma or the mankind, rat or mice serum (to test) or CSF (celiolymph in model system; The mankind, mouse or rat) or human cell's extract in duplex stability:

For example: by dsRNA molecule in 100% human serum (Sigma catalog number (Cat.No.) H4522) with the ultimate density of 7uM at 37 DEG C of incubations.(in human serum, be diluted to the siRNA liquid storage of 100uM with the ratio of 1:14.29, or in the tissue extract from various organization types).Get five microlitres (5ul) and for example, be added to 15ul 1.5xTBE sample-loading buffer at different time point (0,30min, 1h, 3h, 6h, 8h, 10h, 16h and 24h).Sample is freezing in liquid nitrogen immediately, and remain on-20 DEG C.

Each sample is loaded to non-sex change 20% acrylamide gel of preparing according to methods known in the art.Under ultraviolet lamp, show oligomer by ethidium bromide.

exonuclease Stability Determination

In order to study the stabilization of 3 ' non-nucleotide part to nucleic acid molecule, the dsRNA duplex of incubation sense strand, antisense strand and annealing in the endochylema extract of being prepared by different cell types.

Extract: HCT116 endochylema extract (12mg/ml).

Extraction buffer: by before using, newly add 25mM Hepes pH-7.3 (37 DEG C), 8mM MgCl ₂, containing the 150mM NaCl of 1mM DTT.

Method: 3.5ml is mixed with the 46.5ml solution that contains 120mg HCT116 endochylema extract for examination dsRNA (100mM).This 46.5ml solution is made up of 12ml HCT116 extract and 34.5ml Extraction buffer, and this damping fluid has supplemented DTT and protease inhibitor cocktail/100 (Calbiochem, setIII-539134).In incubation pipe, the ultimate density of siRNA is 7mM.By sample at 37oC incubation, and specify time point, get 5ml and move on in new pipe, mix then quick-frozen in liquid nitrogen with 15ml 1XTBE-50% glycerine sample-loading buffer.In sample-loading buffer, the ultimate density of siRNA is 1.75mM (21ng siRNA/ml).For analyzing by native polyacrylamide gel electrophoresis and EtBr dyeing, each swimming lane loading 50ng.Analyze the siRNA of each swimming lane loading 1ng test for Northern.

innate immune response to dsRNA molecule:

Fresh human blood (under room temperature) is mixed by the ratio of 1:1 with the aseptic 0.9%NaCl under room temperature, and then loading (1:2 ratio) arrives on Ficoll (Lymphoprep, Axis-Shield catalog number (Cat.No.) 1114547) gently.By sample under room temperature (22 DEG C, 800g) in bucket type whizzer centrifugal 30 minutes, with the washing of RPMI1640 substratum centrifugal (room temperature, 250g) 10 minutes.To cell counting, and with 1.5x10 ⁶the ultimate density of individual cell/ml is inoculated in growth medium (RPMI1640+10%FBS+2mM L-glutaminate+1%Pen-Strep), and at 37 DEG C, incubation carries out dsRNA processing for 1 hour again.Use Lipofectamine ^tM2000 reagent (Invitrogen) according to the explanation of manufacturers by cell be exposed to different concns for examination dsRNA, and at 37 DEG C at 5%CO ₂incubation 24 hours in incubator.

As the positive control of IFN response, using poly for cell (I:C) (synthetic analogues of a kind of double-stranded RNA as TLR3 part (dsRNA)) (InvivoGen catalog number (Cat.No.) tlrl-pic) with the ultimate density of 0.25-5.0 μ g/mL or the ultimate density processing with 0.075-2 μ g/mL with Thiazolaquinolone (CLO75) (a kind of TLR 7/8 part) (InvivoGen catalog number (Cat.No.) tlrl-c75).To use Lipofectamine ^tMthe cell of 2000 agent treated is as feminine gender (reference) contrast of IFN response.

After incubation, 24 hours time, collecting cell, transfers to supernatant liquor in new pipe.Sample is freezing in liquid nitrogen immediately, use and be used for the DuoSet ELISA test kit (R & D System DY2060) of IL-6 and tested the secretion of IL-6 and TNF-α cytokine according to the explanation of manufacturers for the DuoSet ELISA test kit (R & D System DY210) of TNF-α.From cell precipitation, extract RNA, measured the mRNA level of Human genome IFIT1 (trilateral tetrapeptide repeats interferon inducible protein 1) and MX1 (myxovirus (influenza virus) resistance 1, interferon inducible protein p78) by qPCR.The mRNA amount recording is normalized to the mRNA amount of reference gene peptidyl prolyl isomerase A (cyclophilin A, CycloA).By the amount that derives from the processing IFIT1 of cell and the mRNA of MX1 gene is compared to the induction of having evaluated IFN signal transduction with respect to the amount of its untreated cell.QPCR result is those results of having passed through QC standard, that is, slope of standard curve value is in interval [4 ,-3], and R2>0.99, without primer dimer.The result not requiring by QC is got rid of from analyze.

In general, select, for the dsRNA with particular sequence of vitro test, people and the second species are had to specificity such as rat or rabbit gene.Test the activity of dsRNA to the mankind (Hu), mouse (Ms), rat (Rt), chinchilla (Chn) and cavy (GP) target gene.For example, by clone's chinchilla target gene (being CDKN1B) and 293 or HeLa clone in express and tested activity in chinchilla.Use has these RNA sequences also as the siRNA that has carried out modifying has obtained similar result herein.

PCT/US12/49616 discloses the dsRNA nucleic acid molecule of chemically modified, and entirety is incorporated herein by reference.

embodiment 2: the sequence of the active dsRNA molecule of generation target gene also produces siRNA

Use the known array of the mRNA of proprietary algorithm and target gene disclosed herein, generated the sequence that many potential dsRNA are siRNA.

Specifically, SEQ ID NO:23-381 provides people 19 aggressiveness oligonucleotide; SEQ ID NO:382-693 provides best 19-aggressiveness people across species oligonucleotide; SEQ ID NO:694-1367 provides people 18 aggressiveness oligonucleotide; And SEQ ID NO:16-1495 provides the best 18-aggressiveness people who can be used for generating the dsRNA that lowers HES1 expression across species oligonucleotide; Table I comprises can be used for generating lowers the SEQ ID NO:26 of dsRNA that HES1 expresses, 667-26, shown in 690 based on structure A1 and SEQ ID NO:26,691-26, some the preferred 19 aggressiveness oligonucleotide based on structure A2 shown in 706.

SEQ ID NO:1496-1759 provides people 19 aggressiveness oligonucleotide; SEQ ID NO:1760-2029 provides best 19-aggressiveness people across species oligonucleotide; SEQ ID NO:2030-2575 provides people 18 aggressiveness oligonucleotide; And SEQ ID NO:2576-2703 provides the best 18-aggressiveness people who can be used for generating the dsRNA that lowers HES5 expression across species oligonucleotide; Table II comprises can be used for generating lowers the SEQ ID NO:26 of dsRNA that HES5 expresses, 707-26, shown in 724 based on structure A1 and SEQ ID NO:26,725-26, some the preferred 19 aggressiveness oligonucleotide based on structure A2 shown in 732.

SEQ ID NO:13004-14077 provides people 19 aggressiveness oligonucleotide; SEQ ID NO:14078-14801 provides best 19-aggressiveness people across species oligonucleotide; SEQ ID NO:14802-16389 provides beautiful woman's 18 aggressiveness oligonucleotide; And SEQ ID NO:16390-16621 provides the best 18-aggressiveness people who can be used for generating the dsRNA that lowers HEY2 expression across species oligonucleotide; Table III comprises can be used for generating lowers the SEQ ID NO:26 of dsRNA that HEY2 expresses, 779-26, shown in 784 based on structure A1 and SEQ ID NO:26,785-26, some the preferred 19 aggressiveness oligonucleotide based on structure A2 shown in 788.

SEQ ID NO:7444-8185 provides people 19 aggressiveness oligonucleotide; SEQ ID NO:8186-9007 provides beautiful woman across species oligonucleotide; SEQ ID NO:9008-10233 provides people 18 aggressiveness oligonucleotide; And SEQ ID NO:10234-10533 provides the best 18-aggressiveness people who can be used for generating the dsRNA that lowers CDKN1B expression across species oligonucleotide; Table IV comprises can be used for generating lowers the SEQ ID NO:26 of dsRNA that CDKN1B expresses, 867-26, shown in 886 based on structure A1 and SEQ ID NO:26,887-26, some the preferred 19 aggressiveness oligonucleotide based on structure A2 shown in 900.

SEQ ID NO:16622-18469 provides people 19 aggressiveness oligonucleotide; SEQ ID NO:18470-18643 provides beautiful woman across species oligonucleotide; SEQ ID NO:18644-26211 provides people 18 aggressiveness oligonucleotide; And SEQ ID NO:26212-26666 provides the best 18-aggressiveness people who can be used for generating the dsRNA that lowers NOTCH1 expression across species oligonucleotide; Table V comprises can be used for generating lowers the SEQ ID NO:26 of dsRNA that NOTCH1 expresses, 901-26, shown in 910 based on structure A1 and SEQ ID NO:26,911-26, some the preferred 19 aggressiveness oligonucleotide based on structure A2 shown in 912.

Scoring based on it in proprietary algorithm and determine that the oligonucleotide sequence of priority is the optimum prediction sequence that targeted human genoid is expressed.

" 18+1 " refers to that 19 Nucleotide are long and comprises and the molecule of the mispairing of mRNA target the 1st of antisense strand according to structure A2.In preferred embodiments, sense strand and antisense strand complete complementary.In some embodiments, sense strand is in 1,2 or 3 position and antisense strand mispairing.

embodiment 3: the hitting and miss the target test of double stranded rna molecule:

PsiCHECK ^tMsystem makes it possible to evaluate guiding chain (GS) (antisense) and passerby's chain (PS) (sense strand) causes target (hitting) and the effect of missing the target, and mode is the variation of its target sequence expression level of monitoring.Four are prepared based on psiCHECK ^tMthe construct of-2 (Promega) is to evaluate each activity of the target for examination molecule GS and PS chain and the potential activity of missing the target.In each construct, by a copy of the complete target for examination molecule PS or GS or seed target sequence or the multiple clone site in three copy clone carry sub-downstreams of renilla luciferase translation stop codon in 3'-UTR region.

embodiment 4: the work of combined therapy to the Hair Cell Death that in chinchilla cochlea, carboplatin causes with

Eight chinchillas are carried out to pre-treatment by directly using composition in salt solution of HES1dsRNA, HES5dsRNA and HEY2dsRNA or combination or CDKN1BdsRNA, NOTCH1dsRNA and composition or the combination of HEY2dsRNA in salt solution to the left ear of each animal.The auris dextra that salt solution is administered to each animal is as placebo.Use composition or combine latter two days, carboplatin for animal (75mg/kg abdominal injection) is processed.Putting to death after chinchilla (carboplatin two weeks after treatment), calculating the dead cell per-cent of the middle inner ear hair cells (IRC) of left ear (composition/combined treatment) and auris dextra (brine treatment) and external ear hair cell (ONC).Because acting between dosage of siRNA is similar, therefore the data of 3 kinds of dosage are merged.As previously shown, carboplatin preferentially damages the inner ear hair cells of chinchilla under 75mg/kg dosage, and the maintenance of external ear hair cell is complete.Composition/combination provided in this article has reduced (for example carboplatin causes) inner ear hair cells loss that cochlea middle ear toxin causes.

embodiment 5: the work of combined therapy to the Hair Cell Death that in chinchilla cochlea, the sense of hearing causes with

In chinchilla, study composition of the present invention/the be combined in activity in sense of hearing trauma model.Under 105dB by one group totally 7 animals cause sense of hearing wound by the noise octave band 2.5h being exposed to centered by 4kHz.Composition/combination pre-treatment (48h before sense of hearing wound) of the left ear dsRNA as disclosed herein in salt solution of chinchilla of noise will be exposed to; By vehicle for auris dextra (salt solution) pre-treatment.Complex action potential (CAP) is electrophysiological method reliably for measuring the neururgic convenience that spreads out of from cochlea.CAP carries out record in the following manner: electrode is placed near cochlea base portion, to detect the local field potentials generating in the time that unexpected unlatching sound stimulates such as click or tone burst.After sense of hearing wound, the functional status of every ear is assessed about 2.5 weeks time.Specifically, within 2.5 weeks after sense of hearing wound, determine from the average threshold of the complex action potential of oeil de boeuf record, to determine that whether threshold value in the ear for the treatment of in composition/combination do not treat the threshold value in the ear of (salt solution) lower than (being better than).In addition determine, the amount of inner ear and external ear hair cell loss in ear that composition/combination treats and contrast ear.Result shows that composition provided in this article/combination reduces the ONC loss that in cochlea, sense of hearing wound causes.

embodiment 6: the effect of the Hair Cell Death that combined therapy causes cis-platinum in rat cochlear

Before cisplatin treated, test male Wistar rat to click, 8,16 and the basic auditory brain-stem response (ABR) of 32kHz signal.After basic auditory brain-stem response test, the abdominal cavity transfusion using cis-platinum as 12mg/kg was used through 30 minutes.Treated ear is received in the composition/combination of dsRNA as disclosed herein (being applied directly to round window membrane) in PBS.To contrast the incoherent GFP dsRNA of ear or PBS processes.Within before cis-platinum 3-5 days, use composition/combination to realize protective effect to cochlea using.

Using after cis-platinum 3 days, repeating auditory brain-stem response (ABR).Auditory brainstem response threshold is compared before processing and between after processing, and measure the drift of threshold value.After cisplatin treated, threshold drift is higher shows hair cell loss more serious in cochlea.Repeating after auditory brain-stem response test, by sacrifice of animal, extract cochlea and process by scanning electronic microscope (SEM), the external ear hair cell (ONC) in hook-shaped district (high frequency region) to be lost quantitatively.By cell count disappearance or grievous injury is calculated to external ear hair cell loss per-cent divided by the external ear hair cell sum in the photo visual field.Result shows that composition disclosed herein/be combined in for example to use, when ear toxin (cis-platinum) is used before provides protective effect to cochlea.

embodiment 7: other hearing loss model

a) regeneration of the hearing in cavy (plasticity) model

By single ic injection kantlex (450-500mg/kg) then single iv (jugular vein) injection Ethacrynic Acid (EA) Albino guinea pig carried out to general processing cause deafness.This pharmacological causes is deafly approximately eliminating all hair cells of bilateral after 1-2 days, and leaves the sustenticular cell of differentiation.By through injection of tympanum (TT) or splash into external auditory meatus or ear-drum by ear drop (ErD) and incite somebody to action therapeutic composition/combination as disclosed herein and be applied to middle ear.

Effect of following inspection group compound/combination:

1) using cochlea as carrying out morphological analysis for the whole paving sheet of myosin VIIa (hair cell marker) and Phalloidine dyeing.

2) as the index measurement BrdU combination of hair cell multiplication rate.

b) the acute hearing loss model that noise causes in cavy

Noise can cause the hearing impairment with temporary or permanent sensorineural hearing loss (SNHL) and tinnitus.SNHL can occur individually with tinnitus or together with occur.In the mankind, the hearing loss (NIHL) that noise causes by audiogram, in recruitment phenomenon, in the pathology results of superthreshold hearing test and the threshold drift of the amplitude of otoacoustic emission in declining confirm.Hearing impairment is because being exposed to roughness or pulse noise causes.In addition also should consider, the possibility of pulse noise wound or blast wound.Be exposed to the comparable roughness that is exposed to of pulse noise and cause the more serious pathology of inner ear.The major criterion that noise infringement occurs is sound pressure level (SPL), level gather way (level increase velocity), open-assembly time and individual susceptibility (" vulnerable inner ear ").Post noise exposure causes the threshold value that can partly solve subsequently to raise conventionally, makes transient state component be called " temporary threshold shift " (TTS).Recover if do not existed in the decubation after TTS completely, can cause permanent inner ear infringement (PTS=PTS).Very high sound pressure level can cause mechanical disruption and the PTS of structure in necrocytosis immediately and inner ear.

In this model, induce bilateral pathology by post noise exposure; Cavy is exposed to 117dBSPL broad-band noise 6 hours.

According in the pilot scale research of this model, incite somebody to action composition/combination as disclosed herein for this model, produce useful result.

embodiment 8: the effect of the inner ear ear sensory cell death that combined therapy causes noise

Model system: cavy is exposed to the octave band (130dB SPL) 2 hours (people such as Futon, NeuroReport 19:277-281,2008) centered by 6kHz

Experimental group

The adult Hartley Albino guinea pig (3 monthly age) with normal Preyer is exposed to noise and is divided at random following group: the noise contrast (n=6) of non-processor, use vectorial noise control animal (n=6), animal (n=6) with composition/combination provided in this article with the dosage processing of 1 μ g, animal (n=6) with composition/combination provided in this article with the dosage processing of 5 μ g, animal (n=6) with composition/combination provided in this article with the dosage processing of 10 μ g, animal (n=6) with composition/combination provided in this article with the dosage processing of 50 μ g, adopt the contrast dsRNA compound of lowering EGFP genetic expression respectively with 1 μ g, 5 μ g, 4 groups of noise control animals (every group of n=6) of the dosage processing of 10 μ g and 50 μ g.

Before post noise exposure 1h and after 3 days in process once a day.

Exemplary vehicle: PBS below using in experiment, artificial perilymph.

Composition as herein provided/formulated in combination becomes in experiment vehicle to use.

By vehicle, as herein provided composition/combination or dsRNA control compound through peritoneal injection or inject.Narcotic by lethal dose after functional evaluation is by all sacrifice of animal: every group of three animals were put to death for immune labeled at the 1st day, and remaining animal, at the 21st day, is wherein further processed for scanning electronic microscope (SEM) and analyzes for three.

post noise exposure

By waveform generator (for example: Generator LAG-120B, Leader Electronics Corp, Yokohama, Japan) generate and (for example: A-207R pass through voice amplifier, Pioneer Electronics, Long Beach, California, USA) the continuous pure tone of the 6kHz that amplifies causes noise wound.All animals under anesthesia are exposed to the 6kHz being for example present in, in spacious (: dome tweeter TW340x 0, Audax, Chateau de Loir, France), 120db SPL (sound pressure level) 40min.

the electrophysiologicalmeasurements measurements of auditory function

1h before post noise exposure and after post noise exposure, within 3 days, 7 days and 21 days, measure auditory brain-stem response (ARB).Animal is slightly anaesthetized, and be placed in soundproof room.By three electrodes through subcutaneous insertion right mastoid process (activity), the crown (reference) and left mastoid process (ground connection).For example TDT System 3 of computer-controlled data collecting system (Tucker-Davis Technologies, Alachu, Florida, the USA) data collecting system with Real-time digital signal processing is used for recording ABR and generates acoustic stimuli.Pure tone tone burst (lifting/lowering time, 1ms within the scope of 2 to 24kHz; Total duration, 10ms; Repetition rate, 20/s) be present in spacious with monophonic.Reaction is filtered (0.3-3kHz), digitizing and under the combination of each frequency-sound level, 500 discrete samples averaged.

morphology research: scanning electronic microscope

For example,, as the people such as Sergi B, Protective properties of idebenone in noise-induced hearing loss in the guinea pig.NeuroReport 2006; Described in 17:857 – 861, carry out sem analysis.Letter and easily it, by 2.5% glutaraldehyde perfusion in 0.1M phosphate buffered saline buffer for the cochlea (n=3) of three animals in every group, fixedly spend the night afterwards, then incubation 2h in 2% perosmic anhydride dimethyl arsenic acid buffer liquid.After micro-dissection, by cochlea by concentration from 30% cumulative to 100% ethanol dehydration, and at critical point drying, finally coated with gold.Each sample is observed and taken pictures by for example 50 transmitting SEM equipment of Zeiss Supra (Carl Zeiss Inc., Gottingen, Germany).Carry out the quantitative EM observation of corti's organ configuration of surface by determining the quantity of hair cell in 20 sections (each basilar membrane 1mm is long).If stereocilium bundle does not exist or the stereocilium restrainted merges completely, hair cell is counted to disappearance.The result of hair cell counting is expressed as in every row inner ear hair cells and external ear hair cell to remaining hair cell with respect to the per-cent of the whole length of cochlea.

the dUTP breach end mark assay method of terminal deoxynucleotidyl transferase mediation

The cochlea (n=3) of three animals in every group is passed through to measure reagent (for example Molecular Probes with TUNEL (the dUTP breach end mark of terminal deoxynucleotidyl transferase mediation), Inc., Carslbad, California, USA) dye, as people such as B Sergi, described in Protective properties of idebenone in noise-induced hearing loss in the guinea pig.NeuroReport (2006) 17:857-861.In brief, cochlea is fixing with 10% formaldehyde (pH 7.3) in 0.1M phosphate buffered saline (PBS) (PBS).After micro-dissection, the surperficial prepared product of corti's organ is incubated overnight in ice-cold 70% (v/v) ethanol, then incubation 16h in the DNA marker solution of existing system at room temperature, this label solution contains 10 μ l reaction buffers, 0.75 μ l TdT enzyme, 8.0 μ l BrdUTP and 31.25 μ l dH ₂o.Then tissue is measured to test kit (for example Molecular Probes Inc. with TUNEL, Carlsbad, California, USA) in the anti-BrdU antibody staining (5 μ l antibody+95 μ l corti's organs are at room temperature used to the two 20min of dying of propidium iodide (the 10mM PBS solution of 5 μ g/ml)) of contained Alexa Fluor 488 dye markers.In PBS, after rinsing, corti's organ is for example fixed on, on the slide glass that contains fade-proof mountant (Prolong Gold, Molecular Probes, Inc.).Use confocal laser scanning microscope, CLSM (for example Leica TCS-SP2, Leica Inc., Wetzlar, Germany) to observe sample.

result

The result obtaining in this model shows composition/combination provided in this article: (a) weakened the threshold drift that noise causes; (b) reduced the external ear hair cell loss that noise causes; The protection of the hearing loss (NIHL) that noise is caused is provided.

embodiment 9: combined therapy is regenerated at hair cell in ototoxicity hearing loss rat model in therapeutic activity

Model system.By used 40ul kantlex (200mg/ml) and Ethacrynic Acid (20mg/ml) mixture in PBS (pH 8) through injection of tympanum.6 groups of rats (N=5,4 or 3) are used.

In 1-4 group, by animal (the brown young bull rat of Wistar and Norway (180-220g)) by use through tympanum kantlex as above (KM) and Ethacrynic Acid (EA) mixture combine process and bilateral causes deaf.Animal in the 5th group is as above there being an ear deafness when accepting PBS in picking up the ears.There is not deafness in the animal in the 6th group.By the dsRNA of EGFP with comparing siRNA.Following appointment, will be delivered to 1-4 group for examination or contrast siRNA.The 5th and 6 groups do not accept combination/contrast siRNA treatment.

Carry out the experiment of following group (total n=25):

The 1st group of (5 rats): KM+EA.Left ear: contrast, the combination of auris dextra: HES1dsRNA+HEY2dsRNA+HES5dsRNA;

The 2nd group of (5 rats): KM+EA.Left ear: contrast, auris dextra: the combination of HES1dsRNA+HEY2dsRNA+EGFP dsRNA);

The 3rd group of (5 rats): KM+EA.Left ear: contrast, auris dextra: the combination of NOTCH1dsRNA+CDKN1B dsRNA+HEY2dsRNA;

The 4th group of (4 rats): KM+EA.Left ear: contrast, auris dextra: EGFP dsRNA (contrast siRNA):

The 5th group of (3 rats): KM+EA.Left ear: contrast, auris dextra: vehicle;

The 6th group (3 rats): normal control.Left ear: not operation.Auris dextra: sham-operation (surgical operation)

the details of using of dsRNA combination: as follows, contrast siRNA or dsRNA combination are used by the GelFoam and the pump method of application that combine.

After KM+EA uses the 4th day, contrast siRNA or dsRNA are combined to the animal that is administered to anesthesia via following mode: implement the surgical operation of opening otic vesicle, and on a small pieces GelFoam who is placed on round window membrane, apply the 3 contrast siRNA of μ l volume or the combination of dsRNA (relevant dsRNA heavy dose, referring to table B).Next, the conduit of 2006Alzet miniosmotic pump is placed in to GelFoam with suitable orientation upper, and is fixed to otic vesicle.Then connect the conduit to and be filled with the contrast siRNA of 200 μ l volumes or the pump of dsRNA combination (relevant pump dsRNA dosage, referring to table B), then implantable pump between 2 shoulder blades.Within the time period of 6 weeks, apply continuously contrast siRNA or dsRNA combination via 2006Alzet miniosmotic pump.

Table B.dsRNA dosage

The details of dsRNA compound: table C below provides the details for the chemically modified dsRNA compound of this model system.

Table C

Table D below provides the caption of the modification ribonucleotide/unconventional part of the dsRNA compound for showing C.

Table D: caption

Code	Modify
		Nuc	?
c6Np	Amino modifying agent C6 (Glen Research)
		iB	Inverted deoxidation dealkalize base
mA	2 '-O-methyladenosine-3 '-phosphoric acid ester
		mA$	2 '-O-methyladenosine (without phosphoric acid ester)
mC	2 '-O-methylcytidine-3 '-phosphoric acid ester
		mC$	2 '-O-methylcytidine (without 3 '-phosphoric acid ester)
mG	2 '-O-methylguanosine-3 '-phosphoric acid ester
		mG$	2 '-O-methylguanosine (without phosphoric acid ester)
mU	2 '-O-methyluridine-3 '-phosphoric acid ester
		mU$	2 '-O-methyluridine (without phosphoric acid ester)
rA	Ribose adenosine-3 '-phosphoric acid ester
		rA$	Ribose adenosine (without phosphoric acid ester)
rA2p	Ribose adenosine-2 '-phosphoric acid ester

rC	Ribose cytidine-3 '-phosphoric acid ester
		rC$	Ribose cytidine (without phosphoric acid ester)
rC2p	Ribose cytidine-2 '-phosphoric acid ester
		rG	Ribose guanosine-3 '-phosphoric acid ester
rG2p	Ribose guanosine-2 '-phosphoric acid ester
		rU	Ribose uridine-3 '-phosphoric acid ester
rU$	Ribose uridine (without phosphoric acid ester)
		rU2p	Ribose uridine-2 '-phosphoric acid ester
P	5'-phosphoric acid ester
		z	The prefix of end-blocking part
zc3p	C3Pi covalency is attached
		zc3p$	C3OH covalency is attached
$	Without terminal phosphate ester

ABR/DPOAE measurement setup: for listed all animals in upper table B, using after KM+EA the 3rd day or the 4th day but measured infringement baseline ABR/DPOAE before operation/dsRNA combination/dsRNA contrast of the 4th day is used.For ABR and DPOAE record: ketamine for rat (40mg/kg)-xylazine (5mg/kg) mixture is anaesthetized.

In addition, at the 3rd, 5,7 and 9 weeks to all animal testings ABR/DPOAE.In addition, at the 11st week to all animal testings ABR/DPOAE.

The euthanasia of animal and Histological research: all laboratory animal are being used to 11 weeks enforcement euthanasia (carrying out at the 11st week after ABR/DPOAE test) after dsRNA combination/dsRNA contrast.Gather in the crops inner ear tissue, and accept qPCR research, the RACE analysis of split product etc. of Histological research, target gene and Atoh1mRNA level.

Result: Figure 1A-1E has shown the ABR response obtaining after the 0th day, 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks in this research.Figure 1A shown in this research the 0th day, 1KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.Figure 1B shown in this research the 0th day, 4KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.Fig. 1 C shown in this research the 0th day, 8KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.Fig. 1 D shown in this research the 0th day, 16KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.Fig. 1 E shown in this research the 0th day, 32KHz stimulates after 3 weeks, after 5 weeks, after 7 weeks and the ABR of rear acquisition in 9 weeks responds.Fig. 1 F provides the caption of Fig. 1 A – 1E.

Table E below provides the explanation of the caption of test group in Figure 1A-1E.

Table E: the explanation of the caption of test group in accompanying drawing

Figure 1A-1E has shown in the combination of HES1dsRNA+HES5dsRNA+HEY2dsRNA and the hearing loss model that is combined in this ear toxin-induced of NOTCH1dsRNA+CDKN1B dsRNA+HEY2dsRNA and has effectively obviously improved ABR response.

embodiment 10: the impaired obstacle for the treatment of vestibular function

Can be used for testing animal model that combination disclosed herein, composition and method improve vestibular function and be found in the people such as the Schlecker C that entirety is incorporated herein by reference, Selective atonal gene delivery improves balance function in a mouse model of vestibular disease.Gene Ther.2011 September; 18 (9): 884-90.

Although above embodiment has shown the ad hoc fashion of carrying out embodiment of the present invention, in practice, person of skill in the art will appreciate that the alternative of the execution embodiment of the present invention clearly not illustrating herein.Should be appreciated that the disclosure should be regarded as the example of the principle of the invention, embodiment shown in being not intended to limit the invention to.

Those skilled in the art only use normal experiment method just can recognize the equivalent that maybe can determine particular of the present invention as herein described.This type of equivalent is intended to be contained by following claims.

Claims

1. the combination of a HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor, be used for the progress with prevention, treatment or delay dysaudia, hearing loss and/or disequilibrium for experimenter simultaneously, separately or in order, or prevent ear (sensation) hair cell loss of described experimenter's inner ear.

2. prevent, treat or postpone a progress for experimenter's dysaudia, hearing loss and/or disequilibrium, and/or prevent the method for ear (sensation) hair cell loss of inner ear, comprise and use HES1 inhibitor, HES5 inhibitor and HEY2 inhibitor.

3. combination according to claim 1 and 2 or method, wherein said HES1 inhibitor, described HES5 inhibitor and described HEY2 inhibitor will side by side or in order be used substantially.

4. a composition, the combination that comprises HES1 inhibitor or its pharmacy acceptable salt or prodrug, HES5 inhibitor or its pharmacy acceptable salt or prodrug and HEY2 inhibitor or its pharmacy acceptable salt or prodrug; And pharmaceutically acceptable carrier.

5. according to the combination described in any one in claim 1 to 4, method or composition, wherein every kind of inhibitor is independently selected from the group being made up of little organic molecule, albumen, antibody or its fragment, peptide, plan peptide and nucleic acid molecule.

6. combination according to claim 5, method or composition, wherein every kind of inhibitor comprises nucleic acid molecule or its pharmacy acceptable salt.

7. combination according to claim 6, method or composition, wherein the first nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HES1 gene, the second nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HES5 gene, and the 3rd nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HEY2 gene.

8. combination according to claim 7, method or composition, each of wherein said double chain oligonucleotide comprises the dsRNA molecule with sense strand and antisense strand.

9. according to the combination described in claim 7 or 8, method or composition, wherein connect described nucleic acid molecule, or wherein in RNAistar forms, described nucleic acid molecule is annealed.

10. according to the combination described in any one in claim 7 to 9, method or composition, wherein:

The first double chain oligonucleotide comprises sense strand and antisense strand, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

11. according to combination, method or composition described in any one in claim 1-10, wherein described inhibitor is jointly administered in same preparation to described experimenter.

12. according to combination or method described in any one in claim 1-3 and 5-10, wherein described inhibitor is jointly administered in different preparations to described experimenter.

13. according to combination, method or composition described in any one in claim 1-10, wherein described inhibitor is administered to described experimenter jointly by same approach.

14. according to combination or method described in any one in claim 1-3 or 5-10, wherein described inhibitor is administered to described experimenter jointly by different approach.

15. combinations according to claim 13, method or composition, wherein by described inhibitor by being jointly administered to described experimenter through injection of tympanum.

16. combinations according to claim 13, method or composition, wherein use described inhibitor through part jointly.

17. combinations according to claim 16, method or composition, wherein said topical application comprises to ear-drum and applies ear drop.

18. according to combination or method described in claim 1-3 any one, and wherein said using substantially carried out simultaneously.

19. according to combination or method described in claim 1-3 any one, and wherein said using in order carried out.

20. according to the combination described in any one in claim 7 to 19, method or composition, and wherein at least one double chain oligonucleotide comprises structure (A1) independently:

(A1) 5 ' (N) x – Z, 3 ' (antisense strand)

3 ' Z '-(N ') y – z " 5 ' (sense strand)

Wherein each of N and N ' is can unmodified or adorned ribonucleotide, or is unconventional part;

Wherein each of (N) x and (N ') y is each continuous N wherein or N ' and joins to by covalent linkage the oligonucleotide of next N or N ';

Wherein each of Z and Z ' has an independent existence or does not exist, if but while existence, be that 1-5 the continuous nucleotide, a 1-5 continuous non-nucleotide part of the covalently bound 3 ' end at its existing chain or its combine independently,

Wherein each of x and y is the integer between 18 and 40 independently;

The wherein sequence of (N ') y and (N) the sequence complementation of x; And

Wherein (N) x comprises and the antisense sequences of mRNA complementation of the mRNA of the mRNA that is selected from coding HES1, coding HES5 and the mRNA of coding HEY2.

21. combinations according to claim 20, method or composition, wherein x=y=19 in described double chain oligonucleotide.

22. according to the combination described in any one in claim 7 to 19, method or composition, and wherein at least one double chain oligonucleotide comprises structure (A2) independently:

(A2) 5 ' N ¹-(N) x-Z 3 ' (antisense strand)

3 ' Z '-N ²-(N ') y – z " 5 ' (sense strand)

Wherein N ², N and N ' each be ribonucleotide unmodified or that modify independently, or unconventional part;

Wherein each of x and y is the integer between 17 and 39 independently;

Wherein N ²be covalently bound to (N ') y;

Wherein N ¹be covalently bound to (N) x and be selected from the said target mrna mispairing of HES1mRNA, HES5mRNA and HEY2mRNA or for being selected from the complementary DNA nucleotide segment of mRNA of HES1mRNA, HES5mRNA and HEY2mRNA;

Wherein N ¹for selecting the part of the group that freely uridine, deoxyuridine, ribose thymidine, ribodesose thymidine, adenosine or Desoxyadenosine, dealkalize base ribose part and dealkalize base ribodesose part natural or that modify form;

Wherein z " can exist or not exist, if but while existence, be covalently bound at N ²the end-blocking part of the 5 ' end of-(N ') y; And

Wherein each of Z and Z ' has an independent existence or does not exist, if but while existence, be 1-5 continuous nucleotide, the individual continuous non-nucleotide part of 1-5 or its combination of the covalently bound 3 ' end at its existing chain independently;

Wherein the sequence of (N ') y has complementarity with (N) sequence of x; And wherein the sequence of (N) x has complementarity with the continuous sequence that is selected from the sequence in HES1mRNA (SEQ ID NO:1), HES5mRNA (SEQ ID NO:2) and HEY2mRNA (SEQ ID NO:10).

23. combinations according to claim 22, method or composition, wherein x=y=18 in described double chain oligonucleotide.

24. 1 kinds of commercial packages, comprise according to claim 1 or 3 to combination or composition described in any one in 23.

25. commercial packages according to claim 24, wherein said packing pack is containing label or specification sheets, and it provides some information about the method for the combination described in any one in right to use requirement 1 or 3 to 23 or composition.

26. commercial packages according to claim 25, wherein said label or specification sheets comprise drug administration information.

27. commercial packages according to claim 26, wherein said label or specification sheets comprise using indicates.

28. according to the commercial package described in any one in claim 25 to 27, and wherein said label or specification sheets indicate according to claim 1 or 3 to the combination described in any one in 15 and be applicable to treatment.

29. according to the commercial package described in any one in claim 25 to 27, wherein said label or specification sheets indicate according to claim 1 or 3 to the combination described in any one in 15 or composition and are applicable to prevention, treatment or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent ear (sensation) hair cell loss of inner ear.

The combination of 30. 1 kinds of CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor, for simultaneously, separately or in order for preventing, treat or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent ear (sensation) hair cell loss of inner ear.

The progress of 31. 1 kinds of preventions, treatment or delay experimenter dysaudia, hearing loss and/or disequilibriums, or prevent the method for ear (sensation) hair cell loss of inner ear, comprise and use CDKN1B inhibitor, NOTCH1 inhibitor and HEY2 inhibitor.

32. according to the combination described in claim 30 or 31 or method, and wherein said CDKN1B inhibitor, described NOTCH1 inhibitor and described HEY2 inhibitor will side by side or in order be used substantially.

33. 1 kinds of compositions, comprise at least one CDKN1B inhibitor or its pharmacy acceptable salt or prodrug, at least one NOTCH1 inhibitor or its pharmacy acceptable salt or prodrug and at least one HEY2 inhibitor or its pharmacy acceptable salt or prodrug; And pharmaceutically acceptable carrier.

34. according to combination, method or composition described in claim 30 to 33 any one, and wherein every kind of inhibitor is independently selected from the group being made up of little organic molecule, albumen, antibody or its fragment, peptide, plan peptide and nucleic acid molecule.

35. combinations according to claim 34, method or composition, wherein every kind of inhibitor comprises nucleic acid molecule or its pharmacy acceptable salt.

36. combinations according to claim 35, method or composition, wherein the first nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding CDKN1B gene, the second nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding NOTCH1 gene, and the 3rd nucleic acid molecule is the double chain oligonucleotide in conjunction with the nucleotide sequence of coding HEY2 gene.

37. combinations according to claim 36, method or composition, each of wherein said double chain oligonucleotide comprises the dsRNA molecule with sense strand and antisense strand.

38. according to the combination described in claim 36 or 37, method or composition, wherein connects described nucleic acid molecule, or wherein in RNAistar forms, described nucleic acid molecule is annealed.

39. according to combination, method or composition described in any one in claim 35-38, wherein:

(a) every chain is that 18 to 49 Nucleotide are long independently;

40. according to combination, method or composition described in any one in claim 30-39, wherein described inhibitor is jointly administered in same preparation to described experimenter.

41. according to combination or method described in any one in claim 30-32 and 34-39, wherein described inhibitor is jointly administered in different preparations to described experimenter.

42. according to combination, method or composition described in any one in claim 30-39, wherein described inhibitor is administered to described experimenter jointly by same approach.

43. according to combination or method described in any one in claim 30-32 and 34-39, wherein described inhibitor is administered to described experimenter jointly by different approach.

44. according to the combination described in claim 42 or method, wherein by described inhibitor by being jointly administered to described experimenter through injection of tympanum.

45. according to the combination described in claim 42 or method, wherein described inhibitor is used jointly through part.

46. according to the combination described in claim 45 or method, and wherein said topical application comprises to ear-drum and applies ear drop.

47. according to combination or method described in any one in claim 30-32, and wherein said using substantially carried out simultaneously.

48. according to combination or method described in claim 30-32 any one, and wherein said using in order carried out.

49. according to combination, method or composition described in claim 36 to 48 any one, and wherein at least one double chain oligonucleotide comprises structure (A1) independently:

(A1) 5 ' (N) x – Z, 3 ' (antisense strand)

3 ' Z '-(N ') y – z " 5 ' (sense strand)

Wherein each of x and y is the integer between 18 and 40 independently;

The wherein sequence of (N ') y and (N) the sequence complementation of x; And wherein (N) x comprises the antisense sequences that is selected from the mRNA of mRNA, coding NOTCH1 of coding CDKN1B and the mRNA of the mRNA of coding HEY2.

50. according to the combination described in claim 49, method or composition, wherein x=y=19 in described double chain oligonucleotide.

51. according to the combination described in any one in claim 36 to 48, method or composition, and wherein at least one double chain oligonucleotide comprises structure (A2) independently:

(A2) 5 ' N ¹-(N) x-Z 3 ' (antisense strand)

3 ' Z '-N ²-(N ') y – z " 5 ' (sense strand)

Wherein each of x and y is the integer between 17 and 39 independently;

Wherein N ²be covalently bound to (N ') y;

Wherein N ¹be covalently bound to (N) x and be selected from the said target mrna mispairing of CDKN1B mRNA, NOTCH1mRNA and HEY2mRNA or for being selected from the complementary DNA nucleotide segment of mRNA of CDKN1B mRNA, NOTCH1mRNA and HEY2mRNA;

Wherein the sequence of (N ') y has complementarity with (N) sequence of x; And wherein the sequence of (N) x has complementarity with the continuous sequence that is selected from the sequence in CDKN1B mRNA (SEQ ID NO:7), NOTCH1mRNA (SEQ ID NO:11) and HEY2mRNA (SEQ ID NO:10).

52. according to the combination described in claim 51, method or composition, wherein x=y=18 in described double chain oligonucleotide.

53. 1 kinds of commercial packages, comprise according to composition or combination described in any one in claim 30 to 32 or 34 to 52.

54. according to the commercial package described in claim 53, and wherein said label or specification sheets indicate according to the composition described in any one in claim 30 to 32 or 34 to 52 or combination and be applicable to treatment.

55. according to the commercial package described in claim 53 or 54, wherein said label or specification sheets indicate according to the composition described in any one in claim 30 to 32 or 34 to 52 or combination and are applicable to prevention, treatment or postpone the progress of experimenter's dysaudia, hearing loss and/or disequilibrium, or prevent ear (sensation) hair cell loss of inner ear.